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Bulletin  279  June,  1926   / 

Qlimtterttntt  Agrirultural  lExprrtmrttt  Station 

Nrm  Haunt,  (Smtnrrtiritt 


The  Genetics  and  Morphology  of  Some 
Endosperm  Characters  in  Maize 


P.   C.   MANGELSDORF 


The  Bulletins  of  this  Station  are  mailed   free  to  citizens  of  Connecticut 
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CONNECTICUT  AGRICULTURAL   EXPERIMENT  STATION 

OFFICERS  AND  STAFF 

as  of 
June,  1926 


BOARD  OF  CONTROL 
His  Excellency,  John  H.  Trumbull,  ex-officio,  President. 

Charles  R.  Treat,  Vice  President  Orange 

George  A.  Hopson,  Secretary   Mount  Carmel 

Wm.  L.  Slate,  Jr.,  Treasurer  New  Haven 

Joseph  W.  Alsop    Avon 

Elijah   Rogers    Southington 

Edward  C.   Schneider    Middletown 

Francis  F.  Lincoln   Cheshire 

STAFF. 
E.  H.  Jenkins,  Ph.D.,  Director  Emeritus. 

Administration.  Wm.  L.  Slate,  Jr.,  B.Sc,  Director  and  Treasurer. 

Miss  L.  M.   Brautlecht,  Bookkeeper  and  Librarian. 
Miss  J.  V.   Bekger.  Stenographer  and  Bookkeeper. 
Miss  Mary  E.   Bradley,  Secretary. 
G.  E.  Graham,  In  charge  of  Buildings  and  Grounds. 

Chemistry:  E.  M.  Bailey,  Ph.D.,  Chemist  in  Charge. 

Analytical  C.  E.  Shepard  ~| 

Laboratory.  Owen  L.  Nolan  I    Assistani  Chemists. 

Harry  J.  Fisher,  A.B.       f 
W.  T.  Mathis  J 

Frank  C.  Sheldon,  Laboratory  Assistant. 
V.  L.  Churchill,  Sampling  Agent. 
Miss  Mabel  Bacon,  Stenographer. 

Biochemical  T.  B.  Osborne,  Ph.D.,  Chemist  in  Charge. 

Laboratory.  H.  B.  Vickery,  Ph.D.,  Biochemist. 

Miss  Helen  C.  Cannon,  B.S.,  Dietitian. 

Botany.  G.  P.  Clinton,  Sc.D.,  Botanist  in  Charge. 

E.  M.   Stoddard,  B.S.,  Pomologist. 
Miss  Florence  A.  McCormick,  Ph.D.,  Pathologist. 
Willis  R.  Hunt,  Ph.D.,  Assistant  in  Botany. 

A.  D.   McDonnell,    General  Assistant. 
Mrs.  W.  W.  Kelsey,  Secretary. 

Entomology.  W.  E.  Britton,  Ph.D.,  Entomologist  in  Charge;    State 

Entomologist. 

B.  H.  Walden,  B.Agr.         » 

M.  P.  Zappe,   B.S.  [  Assistant  Entomologists. 

Philip  Garman,  Ph.D.       ) 

Roger  B.  Friend,   B.Sc,   Graduate  Assistant. 

John  T.  Ashworth,  Deputy  in  Charge  of  Gipsy  Moth  Work. 

R.   C.   Botsford,  Deputy  in  Charge  of  Mosquito  Elimination. 

Miss  Grace  A.  Foote,  B.A.,  Secretary. 

Forestry.  Walter  O.  Filley,  Forester  in  Charge. 

H.  W.  Hicock,  M.F.,  Assistant  Forester. 
J.  E.  Riley,  Jr.,  M.F.,  In  charge  of  Blister  Rust  Control. 
Miss  Pauline  A.   Merchant,  Stenographer. 

Plant  Breeding.  Donald  F.  Jones,  S.D.,  Geneticist  in  Charge. 

P.   C.  Mangelsd.orf,   S.D.,  Assistant   Geneticist. 
H.  R.  Murray,  B.S.,  Graduate  Assistant. 

Soil  Research.  M.  F.  Morgan,  M.S.,  Investigator. 

George  D.  Scarseth,  B.S.,  Assistant. 

Tobacco  Sub-station  Paul  J.  Anderson,  Ph.D.,  Pathologist  in  Charge. 

at  Windsor.  N.  T.  Nelson,  Ph.D.,  Plant  Physiologist. 

THE    TUTTLE,    MOREHOUSE    &    TAYLOR    COMPANY 


TABLE  OF  CONTENTS 

PAGE 

Introduction    5*3 

Definitions    Sx4 

Acknowledgments 5*4 

PART  I 

Defective   Seeds 516 

Widespread  Distribution   in   Germplasm 517 

Origin  by   Mutation 518 

Source   of    Material    521 

Simple   Mendelian    Recessives    523 

Crossing  Experiments    523 

Method  of    Crossing    523 

Results  of  Crosses    525 

The  F2  Generation    526 

Summary   of    Crosses    529 

The  Morphology  of  Defective  Seeds   531 

The  Cytologial  Mechanism  of  Endosperm  Formation 531 

Development  After   Fertilization    532 

Development  of  Defective  Seeds   533 

Regular  Development  in   Early  Stages    534 

The    Pericarp    534 

The   Nucellus    534 

The   Endosperm    535 

Starch    Grain    Formation    536 

The  Aleurone   Layer    536 

The  Embryo    537 

General    Aspects 538 

Physiology  of  Defective  Seeds   539 

Rate  of  Growth,  Final  Weight  and  Germination  of  Normal  Seeds  540 
Rate   of    Growth,    Final   Weight  and    Germination   of    Defective 

Seeds 541 

The  Influence  of  Lethal  Factors  in  Heterozygous  Condition   ....  545 

Effect  upon  the  Gametophyte    546 

Linkage    Relations     548 

Linkage  Between  Su  and  de  Factors    550 

Linkage  of   Defectives  with  Each  Other    554 

Linkage  of  Defectives  with  Growth   Factors    558 

A  Plant   Character  for  Defective   Seeds    559 

Discussion     562 

Summary 562 

PART  II 

Non-Hereditary  Defective  Seeds    564 

Parthenocarpic    Defectives 564 

Some    Conditions   Which    Influence   the   Frequency  of    Partheno- 
carpic  Defectives   in   Maize    566 

Influence  of  Age  of  Silks    568 

Influence  of  the  Age  of   Pollen    569 

Arrested  Development    572 

Irregularities  in  the  Fertilization  Mechanism   . .'. 573 

Summary    578 


PART  III 

Genetic  Factors  Which   Influence  the  Texture  of  the  Endo- 
sperm      579 

Brittle    Endosperm    579 

Sugary   x    Brittle    580 

Shrunken    x   Brittle    582 

Brittle  Endosperm  from  Two  Varieties   583 

Shrunken   Endosperm  from  Two   Sources    584 

Waxy  Endosperm  in  China  and  America  584 

Constant  Variation  in  the  Storage  Material  of  the  Endosperm   . .  585 

The   Relative  Development  of  Endosperm  Characters    586 

Discussion     587 

Summary    589 

PART  IV 

Premature   Germination   of   Maize    Seeds    and    Genetic   Factors 

Which   Govern   Dormancy 590 

Complementary    Factors    590 

Description   Summarized    594 

Phenotypical    and    Genetic    Differences    594 

Duplicate    Factors    ; 596 

Ratios  of  8 :  1   and  41  :  1    598 

Summary  of    Breeding"    Behavior    509 

Linkage  Relations 599 

gei  x   Su    600 

ge»  x  su   601 

ge3  and  get,  x  su   601 

Apparent  Linkage  with  Endosperm  Color  Factors    602 

Premature  Digestion  and   Pigment   Formation    603 

The  Relation  of  Premature  Germination  to  Chlorophyll  Develop- 
ment      604 

Physiology  of  Premature  Germination   605 

The  Effects  of  Premature  Germination  on  the  Growth  of  the  Seed  605 

Discussion     607 

Summary    607 

Conclusion    608 

Literature  Cited 610 

Explanation    of   Plates    614 


The  Genetics  and  Morphology  of  Some 
Endosperm  Characters  in  Maize  : 


P.  C.  Mangelsdorf 


Introduction 

When  Nawaschin  in  1898  discovered  the  phenomenon  of  double 
fertilization  in  Lilium  it  was  generally  believed  by  botanists  that 
such  a  peculiar  mechanism  was  confined  to  this  species  and  per- 
haps a  few  closely  related  ones.  Later  investigations  have  shown 
that  it  is  widely  distributed  and  that  the  endosperm  of  angio- 
sperms,  with  perhaps  a  few  exceptions,  is  the  product  of  a  sexual 
fusion,  quite  apart  from  that  which  gives  rise  to  the  embryo,  and 
differing  from  the  latter  in  that  one  male  nucleus  combines  with 
two  or  more  female  nuclei,  while  the  embryo  results  from  a 
fusion  in  which  both  parents  contribute  equally. 

Thus  the  endosperm  of  angiosperms  is  unique  in  several 
respects.  It  resembles  its  near  relative,  the  embryo,  in  its  sexual 
origin,  but  differs  from  the  latter  in  structure,  in  capacity  for 
continued  development,  and  in  ability  to  reproduce. 

This  unique  sporophyte,  if  indeed,  it  may  be  called  a  sporophyte, 
achieves  its  highest  development  in  the  cereals,  in  which  it  con- 
stitutes the  part  which  makes  these  plants  of  such  great  economic 
importance,  and  in  which  it  gives  evidence  of  its  sexual  origin  by 
the  expression  of  the  hereditary  factors  which  it  receives  from  its 
two  parents. 

Mendelian  characters  which  have  their  expression  in  the  endo- 
sperm have  been  found  in  wheat,  rye,  barley,  rice  and  maize. 
They  are  apparently  most  numerous  in  maize  and  a  number  of 
characters  which  affect  the  color  of  the  endosperm  or  aleurone 
layer  and  the  texture  of  the  endosperm  tissue  of  this  species 
have  long  been  familiar,  and  have  played  an  important  part  in 
the  researches  leading  up  to  the  re-discovery  of  Mendel's  Law 


_  *  Parts  I-IV  of  a  thesis  submitted  to  the  Faculty  of  the  Bussey  Institu- 
tion of  Harvard  University  in  partial  fulfillment  of  the  requirements  for 
the  degree  of  Doctor  of  Science,  June,  1925.  Part  V,  on  "Genetic  Factors 
Which  Affect  the  Development  of  the  Gametophyte  and  Their  Relation 
to  Some  Endosperm  Characters,"  has  been  combined  with  researches  by 
Dr.  D.  F.  Jones  on  the  same  subject  and  will  appear  in  Genetics,  Vol.  XI, 
under  the  title  "The  Expression  of  Mendelian  Factors  in  the  Gametophyte 
of  Maize." 


514  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

in  1900  and  in  the  accumulation  of  a  vast  amount  of  genetic 
evidence  since  its  re-discovery. 

The  endosperm  characters  of  maize  are  of  unusual  value  to  the 
geneticist  because,  like  all  endosperm  characters,  they  are  visible 
sooner  than  those  which  affect  other  parts  of  the  plant,  and  because 
they  are  readily  studied  in  large  numbers  without  the  necessity 
of  devoting  a  great  amount  of  land  or  labor  to  the  purpose.  The 
average  ear  of  maize  bears  from  several  hundred  to  a  thousand 
seeds  and  one  pollination  on  a  single  plant  produces  a  large  popu- 
lation which  is  readily  classified  because  the  environment  has 
been  remarkably  alike  for  all  its  members. 

In  recent  years  the  widespread  application  of  a  new  method 
of  corn  improvement  which  involves  the  extensive  inbreeding  of 
this  crop  by  artificial  self-pollination,  has  brought  to  light  many 
new  characters  which  influence  the  development  of  the  endosperm. 
Because  of  their  possible  phylogenetic  significance,  and  because 
they  represent  new  material  which  may  prove  of  value  in  charting 
the  germplasm  of  this  important  species,  these  characters  merit 
a  thorough  study.  The  following  pages  are  devoted  to  the  pre- 
liminary investigations  of  a  number  of  these  new  characters,  their 
breeding  behavior,  morphology,  effect  upon  development,  and 
their  relation  to  each  other  and  to  other  characters. 

DEFINITIONS 

Two  terms  are  used  so  frequently  throughout  the  pages  which 
follow  that  they  deserve  to  be  defined  and  limited. 

The  endosperm  generation  is  the  period  beginning  with  fertil- 
ization and  ending  with  the  disappearance  of  the  endosperm 
through  absorption  or  digestion.  In  the  cereals,  the  endosperm 
persists  until  the  germination  of  the  seed  but  in  some  plants  it  is 
almost  completely  lost  in  the  early  stages  of  development.  This 
period  has  also  been  termed  the  xenia  generation  by  some  writers. 
In  crosses  the  F2  endosperm  generation  is  borne  on  Fx  plants. 

An  endosperm  character  is  any  character  which  has  its  expres- 
sion in  the  endosperm  generation.  The  term  does  not  imply  that 
the  character  in  question  affects  only  the  endosperm,  in  fact  some 
of  the  endosperm  characters  produce  their  major  effect  upon  the 
embryo. 

ACKNOWLEDGMENTS 

Grateful  acknowledgment  is  made  to  Dr.  E.  M.  East,  under 
whose  supervision  this  investigation  was  made  and  these  pages 
written,  for  his  helpful  advice  and  kindly  criticism.  A  word  of 
appreciation  is  due  the  Connecticut  Agricultural  Experiment 
Station  for  an  arrangement  enabling  the  writer  to  complete  these 
studies  while  a  member  of  its  staff,  and  particularly  to  Dr.  D.  F. 


ENDOSPERM    CHARACTERS    IN    MAIZE  5*5 

Jones  for  his  suggestion  of  the  problem,  his  generous  provision  of 
material  including  a  number  of  preliminary  crosses,  and  his  con- 
stant encouragement  throughout.  Acknowledgment  is  also  due 
Dr.  Florence  McCormick  for  advice  in  regard  to  the  preparation  of 
material  for  histological  studies,  to  Helen  Parker  Mangelsdorf, 
for  assistance  in  the  statistical  work,  and  to  numerous  investi- 
gators who  have  contributed  material. 


5*6  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

PART    I 

Defective  Seeds 

Defective  seeds  are  lethal  or  semi-lethal  characters  which  affect 
the  development  of  the  endosperm  and  embryo  between  the  time 
•of  fertilization  and  maturity.  These  characters  were  first  reported 
by  Jones  (1920)  who  described  them  as  "aborted  seeds  with  either 
entirely  empty  pericarps  or  badly  shrivelled  seeds,  completely  lethal 
in  some  cases  and  partially  so  in  others,"  He  found  these  char- 
acters to  be  inherited  as  simple  Mendelian  recessives. 

Previous  to  1920  aborted  seeds  had  frequently  been  noted  on 
open-pollinated  ears  of  maize  but  had  generally  been  regarded  as 
due  to  imperfect  pollination  or  other  external  factors. 

Self-pollinated  ears  in  which  approximately  one  fourth  of  the 
seeds  were  aborted,  had  also  undoubtedly  appeared  in  the  cultures 
of  many  investigators  before  1920  but  these  lethal  characters  were 
not  noted  or  were  not  regarded  as  heritable. 

In  an  early  edition  of  Bailey's  "Plant  Breeding"  appears  a 
photograph  of  two  ears  grown  shortly  after  the  re-discovery  of 
Mendel's  Law,  illustrating  the  alternative  inheritance  of  the 
starchy  and  sugary  conditions  of  the  endosperm.  One  of  these 
ears  is  clearly  segregating  for  defective  seeds  in  addition  to  the 
other  two  characters.  The  segregation  is  so  well  defined  that  the 
normal  and  aborted  seeds  on  three  of  the  rows  of  grain  can  be 
counted  from  the  photograph.  Seventy-five  normal  and  23  defec- 
tive seeds  are  noted.  The  investigator  who  pollinated  these  ears 
to  prove  or  disprove  to  his  own  satisfaction  the  newly  re-dis- 
covered Law  of  Mendel,  had  more  evidence  of  its  correctness 
than  he  probably  realized. 

Richey  (1923)  found  several  defective  seeds  on  an  ear  of  maize, 
believed  to  be  many  centuries  old,  unearthed  from  an  Indian 
graveyard  in  Peru.  He  concludes  from  this  discovery  that  defec- 
tive seeds  are  characters  of  considerable  antiquity.  Although  his 
conclusion  is  probably  correct,  it  is  scarcely  justified  from  this 
evidence  alone,  since  it  is  equally  possible  that  the  few  aborted 
seed  on  this  ancient  ear  are  of  the  non-hereditary  types  described 
in  Part  II. 

In  the  past  few  years  many  experiment  stations  in  this  country, 
Canada,  South  America  and  Europe  have  undertaken  projects  for 
the  improvement  of  corn  by  the  method  outlined  by  East  and 
Jones  (1919)  and  by  Jones  (1920)  and  known  as  "Selection  in 
Self-fertilized  Lines."  Thousands  of  self-pollinations  in  many 
varieties  have  been  made  every  year,  and  this  extensive  inbreeding 
of  a  naturally  cross-fertilized  species  has  brought  to  light  many 
recessive  variations  previously  covered  up  by  the  remarkable 
heterozygosity    which    exists    in    the   average   variety    of    maize. 


ENDOSPERM    CHARACTERS    IN    MAIZE 


5  !  7 


Among  these  variations  have  been  a  large  number  of   defective 
seed  types. 

The  writer  (Mangelsdorf,  1923)  has  noted  defective  seeds  in 
self-pollinated  ears  of  more  than  30  representative  American 
varieties  as  well  as  several  from  Spain,  Italy,  China  and  Peru. 
Since  1920,  defective  seeds  have  been  reported  under  various 
names  by  numerous  investigators.  Lindstrom  (1920,  1923)  has 
described  "abortive,"  "flint  defectives,"  and  "sweet  defectives." 
Demerec  (1923)  has  reported  a  condition  which  he  calls  "germ- 
less,"  Eyster  (1922)  a  peculiar  defect  to  which  he  gives  the  term 


Fig.  51. — Self-pollinated  ears  of  three  New  England  varieties 
which  are  segregating  for  defective  seeds.  The  third,  sixth  and 
seventh  ears  from  the  left  represent  the  original  ears  of  stocks  dei, 
dez  and  de%  respectively. 

"scarred"  and  Wentz  (1924)  a  type  known  as  "miniature  germ." 
Garber  and  Wade  (1924)  report  a  semi-lethal  type  of  defective 
seed  in  their  cultures. 

All  of  these  characters  may  be  considered  as  variations  of  the 
"defective"  condition  since  all  of  them  represent  a  seed  develop- 
ment considerably  below  normal  and  most  of  them  are  lethal  or 
semi-lethal  in  a  homozygous  condition. 


WIDESPREAD  DISTRIBUTION   IN   GERM  PLASM 

Some  conception  of  the  frequency  with  which  defective  seeds-, 
occur  may  be  gained  from  the  following  figures  taken  from  self- 
pollinations  made  in  typical  American  varieties. 


5 18  CONNECTICUT    EXPERIMENT   STATION  BULLETIN    279 

Defective  seeds  were  first  noted  in  a  lot  of  86  self-pollinated 
ears  of  four  New  England  varieties.  Thirteen  of  these  ears,  or 
15.0  per  cent,  were  segregating. 

In  1922,  575  self-pollinated  ears  of  six  regional  strains  of 
Sanf ord  White  Flint  were  examined  for  these  variations.  Nine- 
teen of  these  ears,  or  3.3  per  cent,  were  found  to  be  segregating. 

Hutchison  (1922),  in  making  a  systematic  search  for  variations 
of  all  sorts,  self-pollinated  2,110  ears  representing  468  different 
lots  of  seed  which  had  been  obtained  from  seed  companies  and 
experiment  stations.  These  lots  contained  most  of  the  varieties 
commonly  grown  in  the  Northern  states  and  included  sweet,  pop, 
dent,  and  flint  types.  Sixty-seven  of  these  ears,  or  3.2  per  cent, 
were  found  to  be  segregating  for  defective  seeds. 

The  percentage  of  segregating  ears,  following  the  first  self- 
pollination,  in  the  lot  of  575  ears  of  Sanford  White,  agrees  so 
closely  with  the  percentage  found  in  Hutchison's  2,110  ears,  that 
these  figures,  3.3  and  3.2,  probably  represent  the  average  condi- 
tion of  most  varieties  of  maize.  In  other  words,  about  one  plant 
in  every  30  in  the  average  variety  is  heterozygous .  for  a  lethal 
factor  which  causes  defective  seeds.  In  some  varieties  this  pro- 
portion is  probably  higher,  depending  to  some  extent  on  the 
amount  of  natural  self-pollination  which  has  occurred  in  past 
generations. 

ORIGIN  BY   MUTATION 

The  relatively  high  frequency  of  these  lethal  characters  in  most 
varieties  suggests  that  maize,  like  Drosophila,  is  constantly  under- 
going factor  changes  at  various  points  in  the  germplasm  and  that 
a  large  proportion  of  these  changes  may  be  lethal  in  their  effect. 
Muller  and  Altenburg  (1919),  in  a  study  to  determine  the  fre- 
quency of  mutation  in  the  X  chromosome  of  Drosophila,  find  that 
characters  which  are  lethal  or  semi-lethal  are  the  most  frequent 
to  occur.  They  estimate  that  the  X  chromosome  in  Drosophila 
produces  a  new  lethal,  on  the  average,  about  once  in  every  100 
generations.  Recent  evidence  from  homozygous  inbred  strains 
of  maize  indicates  that  the  frequency  of  lethal  mutations  in  this 
species  may  be  as  high  or  perhaps  considerably  higher. 

In  1 92 1  an  inbred  strain  of  maize  which  had  been  self -polli- 
nated for  thirteen  generations  and  which  was  apparently  homozy- 
gous for  its  genetic  factors,  as  demonstrated  by  a  test  made  by 
Jones  (1924),  began  to  segregate  for  defective  seeds.  The  sud- 
den appearance  of  this  new  character  was  clearly  due  to  a  germ- 
inal change  since  nothing  of  this  kind  had  previously  been  noted, 
although  a  careful  search  for  new  variations  in  all  inbred  strains 
had  been  constantly  maintained.  Nor  could  this  new  character 
have  been  the  result  of  a  segregation  following  accidental  cross- 
ing since  outcrossing  with  unrelated  stocks  is  immediately  apparent 


ENDOSPERM    CHARACTERS    IN    MAIZE 


519 


by  the  increased  vigor  and  productiveness  of  the  hybrid  plants. 
With  the  exception  of  the  segregation  for  defective  seeds,  the 
mutant  stock  differed  in  no  detail  from  the  original  inbred  strain. 
This  new  character  originating  by  mutation  in  a  homozygous 
stock  is  a  typical  defective  seed,  is  completely  lethal  in  its  effect 


Fig.  52.— Ears  of  a  strain  of  Learning  which  mutated  to  defective 
seeds  after  thirteen  generations  of  inbreeding.  The  ear  at  the  right 
is  segregating  for  the  mutant  character. 


and  is  inherited  as  a  simple  Mendelian  recessive.  Ears  of  this 
inbred  strain  which  are  segregating  for  defective  seeds  are  shown 
in  Fig;  52. 

A  sister  strain  separated  from  this  one  after  seven  generations 
of  inbreeding  also  showed  defective  seeds  in  the  thirteenth  genera- 
tion when  grown  by  H.  A.  Wallace  at  Des  Moines,  Iowa.     This, 


520  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    2/9 

same  strain,  though  of  slightly  different  pedigree,  produced  defec- 
tive seeds  on  two  ears  in  1924  after  17  generations  of  inbreeding. 
The  parental  ears  of  both  segregating  progenies  were  normal  in 
1923  so  two  separate  mutations  must  have  occurred.  In  other 
words,  four  separate  mutations  have  appeared  in  the  germplasm 
of  this  strain  in  the  past  four  years  and  each  time  a  lethal  char- 
acter, defective  seeds,  resulted.  The  pedigree,  showing  the  genera- 
tions in  which  the  mutations  were  first  noted,  is  given  below : 

T-I-I-I-I-K^1)5^-   ^ 
4-4-2-1   <      (l)  >(2)  seg.  de. 

1 -6- 1 -3-4-4-4-2  <^  3  K1)  se&»-  ae- 

^5-5-2-i-i-(i)  seg.  de. 


The  two  defectives  which  were  noted  in  1924  are  similar  in 
appearance  but  both  are  quite  different  from  the  one  found  in 
1921.  Though  no  crosses  have  yet  been  made  between  these 
four  separate  mutations,  it  is  certain  that  they  are  of  at  least  two 
distinct  types  phenotypically  and  probably  genetic  differences  will 
be  found  as  well. 

In  the  past  four  years  only  a  few  ears  have  been  self-pollinated 
each  season  from  this  strain.  The  fact  that  four  separate  muta- 
tions have  been  noted  in  this  rather  small  sample  indicates  that 
germinal  changes  are  now  occurring  rather  frequently.  Previous 
to  1921,  however,  no  mutations  in  this  stock  had  ever  been  noted, 
though  an  active  search  for  variations  was  constantly  maintained 
and  in  some  seasons  a  large  number  of  ears  were  self-pollinated. 
The  only  other  mutations  ever  found  in  inbred  strains  have  been 
red  cobs  in  a  white  cob  strain,  dwarf  plants  in  another  strain  and 
a  chlorophyll  deficiency  in  a  third. 

From  the  limited  experience  with  these  long-inbred  and  rela- 
tively homozygous  strains  of  maize  it  is  evident  that  germinal 
changes  do  occur  and,  perhaps,  very  frequently.  As  in  Droso- 
phila,  a  high  proportion  of  these  changes  probably  result  in  lethal 
factors.  The  dominant  lethals,  if  they  occur  at  all,  are  imme- 
diately lost,  because  individuals  which  carry  them  do  not  live  to 
reproduce.  The  recessive  factors,  unless  they  have  a  marked 
deleterious  effect  in  the  heterozygous  condition,  may  be  carried 
along  for  generations.  It  is  not  at  all  surprising,  therefore,  to 
find  these  lethal  factors  in  almost  every  variety  of  maize.  In- 
breeding brings  them  to  light  and  demonstrates  that  about  one 
plant  in  every  thirty  is  heterozygous  for  one  or  more  of  them. 
How  many  genetically  distinct  lethal  seed  factors  there  are  in 
maize  can  not  be  estimated  at  the  present  time,  but  some  indica- 
tion of  the  enormous  number  which  probably  exists  may  be  gained 
from  the  following  pages. 


ENDOSPERM    CHARACTERS    IN    MAIZE  .  52  1 

SOURCE  OF  MATERIAL 

As  already  mentioned,  defective  seeds  were  noted  by  the  writer 
in  more  than  30  varieties  of  maize.  It  soon  became  evident,  that 
in  order  to  make  a  thorough  investigation  it  would  be  impossible 
to  study  the  breeding  behavior  of  more  than  half  this  number. 
Accordingly  only  those  stocks  in  which  defective  seeds  had  appeared 
at  least  two  successive  generations,  and  in  which  the  segregating 
ears  gave  clear-cut  3:1  ratios,  were  continued.  Fourteen  stocks 
met  these  requirements  and  were  retained. 

It  is  almost  certain  that,  in  confining  the  investigation  to  those 
strains  in  which  simple  3  :i  ratios  were  obtained,  lethals  which  are 
due  to  duplicate  or  triplicate  factors  were  eliminated.  It  was  con- 
sidered best,  however,  to  study  first  the  inheritance  of  the  simple 
recessives  without  the  complications  brought  in  by  duplicate  or 
triplicate  factors,  especially  since  defectives  which  segregate  in 
15:1  or  63:1  ratios  would  be  difficult  to  distinguish  from  the 
various  types  of  non-hereditary  defectives  which  occur  in  small 
numbers  on  almost  every  ear  and  which  are  discussed  in  some 
detail  in  Part  II. 

The  source  of  the  fourteen  types  of  defective  seeds  used  in  this 
investigation  is  given  below.  The  de  numbers  under  which  they 
are  discussed  were  assigned  after  all  the  types  had  been  arranged 
in  a  series  on  the  basis  of  their  "defectiveness."  Those  with  high 
numbers  such  as  de13  and  de14  are  the  most  defective  in  appear- 
ance, while  those  with  low  numbers,  such  as  de1}  de2,  and  de3  most 
nearly  approach  the  normal  condition  and  are  only  semi-lethal  in 
effect,  being  sometimes  obtained  in  a  homozygous  condition. 

dex 

This  type  appeared  following  the  first  self-pollination  of 
Gold  Nugget,  an  eight  rowed,  large-eared,  yellow,  flint  variety. 
Twenty  self-pollinated  ears  were  obtained  of  which  three  were 
segregating  for  defective  seeds.  Only  one  strain  has  been  kept 
heterozygous  for  defective  seeds. 

de2 

This  defective  was  obtained  from  a  self-pollinated  ear  of  an 
eight-rowed  yellow  flint  type  grown  by  Dr.  E.  G.  Anderson,  then 
at  Cornell  University.  This  is  probably  the  same  stock  in  which 
Lindstrom  found  his  "flint  defective"  and,  if  so,  was  obtained 
originally  from  Dr.  W.  E.  Castle  of  Harvard  University. 

dez 

Defective  seeds  appeared  on  a  self-pollinated  ear  of  Century 
Dent,  a  many-rowed,  medium  early,  yellow,  New  England  dent 
variety.     Five  ears  out  of  18  which  were  self -pollinated,  segre- 


52  2  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

gated  for  defective  seed,  but  only  this  and  one  other,  de&,  have 
been  continued. 

de± 

This  type  was  first  noted  in  the  third  generation  of  inbreeding 
in  two  ears  of  a  strain  of  Beardsley's  Learning,  a  many-rowed, 
fairly  late,  yellow  dent  variety.  No  defectives  had  been  noted 
in  this  strain  in  the  first  two  generations  and  their  appearance  in 
the  third  may  have  been  due  to  mutation. 

de5 

This  stock  originated  from  a  segregating  self-pollinated  ear 
of  Reid's  Yellow  Dent  received  from  Dr.  J.  R.  Holbert  of 
Bloomington,  Illinois. 

deQ 

A  self-pollinated  ear  of  Luce's  Favorite,  a  large  eared,  New 
England  dent  variety  received  from  Dr.  R.  A.  Emerson,  segre- 
gated for  defective  seeds  of  this  type. 

de7 

A  mutation  in  an  inbred  strain  of  Chester's  Learning  which  had 
been  self-pollinated  for  thirteen  successive  generations  and  was 
apparently  homozygous,  gave  rise  to  this  defective. 

de8 

This  stock  originated  from  another  ear  of  the  same  lot  of 
self-pollinated  ears  of  Century  Dent  as  dez. 

dea 

This  type  was  found  on  a  self -pollinated  ear  of  Cornell  No.  12, 
a  selection  of  Funk's  90  Day,  obtained  from  Dr.  R.  A.  Emerson. 

de10 

This  defective  appeared  in  a  stock  of  "fine  striped"  which  had 
been  obtained  some  years  previously  from  Cornell  University. 

dexx 

The  source  of  this  stock  was  a  self-pollinated  ear  of  Clarage 
Dent,  a  typical  Western  yellow  dent  variety  received  from 
Professor  M.  T.  Meyers,  Ohio  University. 

de12 

Seeds  of  this  type  appeared  in  a  self-pollinated  ear  of  Burbank's 
"Rainbow,"  a  novelty  purchased  from  Peter  Henderson  Co., 
New  York. 


ENDOSPERM    CHARACTERS    IN    MAIZE  523 

de13 

This  type  was  isolated  from  a  cross  made  by  Mr.  H.  A.  Wallace 
of  Des  Moines,  Iowa.  The  seed  parent  was  a  hybrid  combination 
of  four  inbred  strains ;  the  pollen  parent  a  plant  of  "Illinois  Two 
Ear"  which  Mr.  Wallace  believed  to  be  homozygous  for  defective 
seeds. 

de14: 

This  defective  appeared  in  the  second  generation  of  inbreeding 
of  a  strain  of  Beardsley's  Learning,  the  same  variety  which  gave 
rise  to  de±.  Defective  seeds  were  not  noted  in  this  strain  in  the 
first  generation  of  inbreeding. 

SIMPLE    MENDELIAN   RECESSIVES 

Except  for  the  fact  that  there  is  often  a  slight  deficiency  of 
recessives,  whereas  an  excess  might  be  expected  because  of  the 
regular  occurrence  of  non-hereditary  defectives  on  almost  every 
ear,  all  of  these  fourteen  types  appear  to  be  inherited  as  simple 
Mendelian  recessives. 

CROSSING    EXPERIMENTS 

The  first  defectives  studied,  det,  de2,  dez,  and  de8)  showed  slight 
phenotypical  differences,  the  first  three  being  "partial"  defectives ; 
the  last  a  "complete"  defective.  Crosses  of  these  four  strains 
made  by  Dr.  D.  F.  Jones,  the  later  generations  of  which  were 
classified  by  the  writer,  indicated  that  these  four  defectives  were 
genetically  distinct.  The  next  step  was  to  determine  the  number 
of  factors  involved  in  the  remaining  ten  stocks. 

METHOD  OF   CROSSING 

Throughout  the  investigation  the  general  method  of  crossing 
two  stocks  has  been  as  follows :  A  number  of  tassels,  five  or 
more,  of  the  strain  to  be  used  as  pollen  parent  were  bagged. 
When  pollinations  were  made  the  pollen  from  all  of  the  bagged 
plants  was  collected,  combined  and  mixed.  Theoretically  two- 
thirds  of  the  plants  in  any  segregating  stock  are  heterozygous  for 
the  lethal  factor  and  one-third  are  homozygous  for  the  dominant 
allelomorph.  Half  of  the  pollen  grains  of  the  heterozygous  plants 
should  carry  the  lethal  factor  while  the  remaining  half,  as  well 
as  all  of  the  pollen  from  the  homozygous  plants,  should  lack  the 
lethal  factor.  Assuming  an  equal  production  of  pollen  by  the 
heterozygous  and  homozygous  plants,  a  composite  collection  of 
pollen  made  in  this  way  should  contain,  on  the  average,  one-third 
of  the  pollen  grains  carrying  the  lethal  factor  and  two-thirds 
carrying  its  dominant  allelomorph. 


524  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    2/9 

The  seed  parent  of  the  cross  should,  like  the  pollen  parent,  pro- 
duce heterozygous  and  homozygous  plants  in  the  proportion  of 
2  :i.  When  the  composite  mixture  of  pollen  is  applied  to  homozy- 
gous plants,  only  normal  seeds  should  be  produced.  When 
applied  to  the  heterozygous  plants,  of  which  half  the  ovules  carry 
the  lethal  factor,  one-sixth  of  the  seeds  should  be  defective  if  the 
two  stocks  which  are  crossed  are  alike  in  their  lethal  factors,  but 
all  of  the  seeds  should  be  normal  if  the  lethal  factors  of  the  two 
parental  stocks  are  unlike. 

Since  on  the  average  two-thirds  of  the  plants  of  a  segregating 
stock  are  heterozygous  for  the  lethal  factor,  the  odds  against 
obtaining  no  heterozygous  plants  when  pollinations  are  made  by 
this  method  are  as  follows  : 


^0.    Ears  P 

jllinated 

Odds 

I 

2:1 

2 

8:i 

3 

26:1 

4 

8o:i 

5 

242:1 

In  order  to  be  reasonably  certain  of  including  at  least  one 
heterozygous  plant  in  every  cross  and  to  allow  for  failure  to 
secure  seed,  it  was  customary  to  pollinate  five  ears.  The  pollen 
was  always  collected  from  five  or  more  plants  and  it  is  practically 
certain  that  some  pollen  from  heterozygous  plants  was  always 
included  in  the  mixtures. 

This  method  of  making  the  crosses  between  stocks,  rather  than 
between  individual  plants,  has  the  advantage  of  being  very  rapid, 
a  large  number  of  pollinations  being  made  from  a  single  collec- 
tion of  pollen. 

In  several  cases  in  which  crosses  were  made  between  strains 
which  regularly  bear  two  ears,  one  of  the  ears  was  self-fertilized 
to  determine  the  composition  of  the  plant,  the  other  was  crossed. 
At  harvest,  only  the  crosses  between  known  heterozygotes  were 
retained.  This  method  requires  so  much  additional  time  and 
labor  that  its  possible  advantages  are  offset  by  the  fact  that  only 
a  limited  number  of  crosses  can  be  made  in  a  season.  The  same 
objections  were  found  to  the  method  used  by  Demerec  (1923) 
who,  in  making  crosses  between  white  seedling  stocks,  pollinated 
the  ears  with  a  mixture  of  own  and  foreign  pollen  and  separated 
the  selfed  and  crossed  seeds  by  the  effects  of  xenia.  Only  stocks 
which  differ  in  their  endosperm  color  or  texture  can  be  crossed 
by  this  method. 

That  the  method  of  crossing  at  random  without  determining  the 
composition  of  the  plants  used,  gives  the  results  which  are  theo- 
retically expected,  is  shown  by  the  following  experiment,  which 
incidentally  shows  that  these  lethal  factors   retain  their   srenetic 


ENDOSPERM    CHARACTERS    IN    MAIZE  525 

identity  as  do  any  other  Mendelian  characters.  A  stock  in  which 
defective  seeds  had  appeared  for  three  successive  generations  was 
crossed  with  another  stock  originally  from  the  same  source,  but 
which  had  been  crossed  with  an  entirely  unrelated  strain  and  the 
defective  seeds  recovered  in  the  second  generation.  The  pedigree 
of  these  two  stocks  is  shown  below  : 

>i-i  =  Stock  A 
105-9-7  \Cross-9-1  =  Stock  B 

Six  ears  of  Stock  A  were  pollinated  by  a  mixture  of  pollen 
collected  from  six  plants  of  B.  Four  of  the  six  ears  proved  to  be 
segregating  for  defective  seeds.  A  count  of  the  normal  and 
defective  seeds  on  these  four  segregating  ears  is  shown  in  Table  I. 


Table  i.     Ratios  Resulting  when  Plants  Heterozygous  for  a  Lethal  Factor 

are  Pollinated  with  a  Composite  Mixture  of  Pollen  from 

Homozygous  and  Heterozygous  Plants. 


ar  No. 

Normal 

Defective 

448 
449 
450 
451 

184 
Il6 

ISI 
164 

22 
32 
32 
43 

Total 

615 

129 

Ex.  5  :i 
Deviation 

620 

5 

124 

The  agreement  of  the  actual  results  with  the  theoretical  expec- 
tation is  surprisingly  good.  Exactly  two-thirds  of  the  ears  proved 
to  be  segregating  and  exactly  one-sixth  of  the  seeds  on  these  ears 
were  defective. 

RESULTS  OF  CROSSES 

It  was  believed  that  the  most  rapid  progress  in  determining  the 
total  number  of  lethals  involved  in  the  fourteen  stocks,  would  be 
made  by  crossing  the  first  four,  which  were  apparently  all  differ- 
ent, with  the  remaining  ten,  on  the  assumption  that  some  of  the 
untested  stocks  would  prove  to  be  carrying  the  same  lethal  factors 
as  the  first  four  and  these  could  then  be  eliminated  from  further 
investigation. 

Thirty-nine  crosses  were  made  in  1922  with  the  astonishing 
results  that  the  Ft  seeds  were  normal  in  every  case.  This  indi- 
cated that  not  one  of  the  ten  stocks  carried  the  same  genetic 
factors  for  defective  seeds  as  the  four  original  strains  by  which 
they  had  been  crossed, 


526  CONNECTICUT   EXPERIMENT    STATION  BULLETIN    279 

The  next  step  was  to  cross  the  stocks  in  all  combinations  among 

themselves.     This  program  involved  a  total  of  91  crosses  

of  which  45  had  already  been  made.  Since  the  remainder  could 
not  all  be  made  in  a  single  season,  it  was  decided  to  cross  first 
only  the  stocks  in  which  the  defective  seeds  showed  some'  resem- 
blance. In  appearance  the  fourteen  types  range  from  complete 
defectives  in  which  the  caryopsis  consists  of  little  more  than  the 
flattened,  transparent,  pericarp  to  the  partial  defectives  in  which 
the  recessive  seeds  are  about  half  the  size  of  normal  seeds. 
Between  these  two  extremes  are  all  gradations  and  within  each 
type  there  is  a  certain  amount  of  variation.  Several  representa- 
tive types  of  defectives  are  shown  in  Fig.  53.  The  fourteen  types 
were  arranged  in  a  series  on  the  basis  of  the  average  appearance 
of  the  recessive  seeds.  The  plan  was  to  cross  each  type  with  the 
two  or  three  others  nearest  to  it  in  the  series.  Although  it  was 
realized  that  resemblance  in  appearance  did  not  necessarily  mean 
genetic  relationship,  it  seemed  only  reasonable  to  suppose  that 
types  resembling  each  other  phenotypically  were  more  likely  to  be 
alike  genetically  than  those  which  were  wholly  different  in 
appearance. 

Twenty-seven  crosses  between  types  close  together  in  the  series 
were  made  in  1923  and  again  every  cross  produced  only  normal 
seeds. 

Four  additional  crosses  were  made  in  1924  and  eight  more  in 
1925;  these,  with  the  six  preliminary  crosses  of  1920  and  1921, 
bring  the  total  number  to  84.  Two  of  the  crosses  were  recipro- 
cals, however,  so  that  the  actual  number  of  distinct  combinations 
is  only  82.  This  leaves  9  of  the  possible  91  crosses  which  are  not 
yet  made. 

Of  all  these  crosses,  only  one,  a  combination  of  de5  and  dellf 
gave  defective  seeds  in  Fa.  This  shows  that  de5  and  delt  are 
genetically  identical,  and  that  the  results  of  any  crosses  made  with 
one  of  these  stocks  applies  as  well  to  the  other.  Taking  this  fact 
into  consideration  only  seven  combinations  remain  to  be  made. 

THE  F2  GENERATION 

In  order  to  be  certain  that  segregating  plants  had  always  been 
included  in  making  the  crosses,  and  to  obtain  additional  evidence 
that  the  two  types  entering  the  cross  were  genetically  distinct  in 
each  case,  F2  progenies  of  a  large  proportion  of  the  crosses  have 
been  grown. 

Since  the  heterozygous  crossed  ears  could  not  be  distinguished 
from  the  homozygous  ones,  all  of  the  ears  of  a  cross  were  com- 
bined by  counting  off  an  equal  number  of  seeds  from  each.     As 


ENDOSPERM    CHARACTERS    IN    MAIZE 


527 


one-third  of  the  pollen  grains  of  a  composite  collection  of  pollen 
are  expected  to  carry  the  lethal  factor  of  the  pollen  parent,  and 


Fig.  53. — Six  types  of  defective  seeds  showing  the  gradation  from 
complete  to  partial  defectives.  Normal  seeds  from  same  ears  are 
shown  at  left. 


one-third  of  the  ovules  on  a  composite  collection  of  ears  the  lethal 
factor  of  the  seed  parent,  then  a  mixture  of  seed  obtained  from 


528  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

such  crosses  would  be  expected  to  give  on  the  average  the  fol- 
lowing F2  progenies : 

4  Ears  not  segregating. 

2  Ears  segregating  for  defective  of  pollen  parent. 
2  Ears  segregating  for  defective  of  seed  parent. 
1  Ear  segregating  for  both  defectives. 

The  di-hybrid  ears  are  expected  only  once  in  nine  times.  How- 
ever, if  the  defectives  contributed  by  the  two  parents  show  slight 
phenotypical  difference,  then  the  reappearance  of  two  distinct 
types  in  F2  may  be  regarded  as  fairly  conclusive  evidence  that 
heterozygous  plants  of  both  parents  were  included  in  making  the 
cross  and  that  the  parental  types  are  therefore  genetically  unlike. 

In  order  to  have  better  than  an  even  chance  of  obtaining  di- 
hybrid  ears,  it  was  customary  to  self -pollinate  fifteen  to  twenty 
plants  of  each  cross.  Though  this  method  of  making  crosses  in 
a  wholesale  manner  and  self -pollinating  F2  progenies  on  the  same 
prodigious  scale,  may  appear  to  entail  unnecessary  labor,  in 
reality,  it  proved  to  be  the  most  economical  procedure.  It  is  true 
that  by  growing  only  the  crosses  between  plants  known  to  be 
heterozygous,  di-hybrid  ears  would  be  expected  once  out  of  every 
four  trials  instead  of  once  in  every  nine.  Self-pollinating  vigorous 
Fx  plants  on  a  large  scale  can  be  done  very  rapidly,  however,  and 
it  has  been  found  easier  to  make  the  crosses  at  random  and 
pollinate  twice  as  many  F2  progenies,  than  to  make  individual 
crosses  between  numbered  plants  and  self-pollinate  fewer  F2 
progenies. 

A  total  of  1089  F2  progenies  of  crosses  made  by  this  method 
have  been  self -pollinated.  The  ratio  of  non-segregating  to  mono- 
hybrid  and  di-hybrid  ears  is  given  in  Table  2. 


Table  2.     Non-segregating,    Mono-hybrid,    and    Di-hybrid    Progenies 
Obtained  in  F2  from  Crosses  Made  at  Random. 

Found 

Ears  not  segregating 428 

Ears  segregating  one  type 552 

Ears  segregating  both  types  109 

The  number  of  dy-hybrid  ears  obtained  agrees  very  closely  with 
the  theoretical  expectation.  There  is  a  significant  excess  of  the 
mono-hybrid  ears,  however.  These  are  expected  only  as  fre- 
quently as  the  non-segregating  ears,  actually  they  have  appeared 
in  considerable  excess.  A  greater  production  of  pollen  by  the 
heterozygous  plants  in  some  of  the  stocks,  or  the  occurrence  of 
heterozygous  plants  more  frequently  than  two  out  of  three,  might 
account  for  this. 


Expected 

Deviation 

484 
484 

-56 

68 

121 

— 12 

ENDOSPERM    CHARACTERS    IN    MAIZE 


529 


SUMMARY  OF  CROSSES 


The  diagram  in  Fig.  54  gives  a  picture  of  the  situation  with 
respect  to  these  fourteen  stocks.*  Squares  with  vertical  cross 
hatching  represent  crosses  in  which  the  Fx  seeds  were  normal. 
Those  with  horizontal  cross  hatching  represent  crosses  in  which 


dej        dej        de4        des         de6        *07        de8        deg       <Je10      le^      <Sei2      de13      &*u 


I  P,   Seeds  Befectlve 


>,    Seeds   Normal 


Two  Types   in  Fg 


Dl-hybrid  Ears   in  P2 


Fig.  54.— Diagram  showing  the  crosses  which  have  been  made  among 
the  fourteen  defective  seed  stocks.  The  types  were  arranged  in  the  order 
of  their  "defectiveness." 

the  Fx  seeds  were  normal  and  two  distinct  types  of  defectives 
appeared  in  F2  though  no  di-hybrid  ears  were  obtained.  Squares 
with  diagonal  cross  hatching  represent  crosses  in  which  the  F± 

*  For  reasons  of  economy  the  detailed  data  showing  the  segregation  in 
the  individual  ears  of  the  fourteen  parental  stocks  and  their  crosses  are 
not  included.  Any  marked  deviations  from  expectation  are  noted,  however, 
and  are  discussed  in  this  and  other  papers. 


53°  CONNECTICUT   EXPERIMENT    STATION  BULLETIN   279 

seeds  were  normal  and  one  or  more  di-hybrid  ears  were  obtained 
in  F2,  while  the  single  case  in  which  defective  seeds  appeared  in 
Fx  is  shown  by  a  solid  square. 

As  has  already  been  pointed  out,  the  production  of  only 
normal  seeds  in  Ft  is  fairly  good  evidence  that  the  two  types 
entering  the  cross  are  genetically  distinct  providing  that  three  or 
more  ears  have  been  crossed.  The  reappearance  of  two  distinct 
types  in  F2  is  still  better  evidence,  while  the  occurrence  of  one  or 
more  di-hybrid  ears  may  be  safely  regarded  as  conclusive  proof. 

The  squares  are  numbered  diagonally  from  top  to  bottom  and 
from  left  to  right.  Thus  1-14  represent  self-pollinations:  15-27 
the  crosses  between  types  immediately  adjacent  in  the  series ; 
28-39  crosses  between  types  one  degree  apart  in  the  series  and 
40-50  crosses  between  types  two  steps  apart.  In  other  words 
the  crosses  15-50  represent  the  most  important  combinations.  It 
will  be  noted  that  all  of  these  36  crosses  have  been  made.  In  27 
of  these  combinations  di-hybrid  ears  have  been  obtained  in  F2. 
In  five  crosses,  Nos.  18,  t,7>  4°>  43>  and  46,  two  distinct  types  of 
defectives  reappeared  in  F2,  while  three  of  the  combinations, 
Nos.  21,  33  and  41,  have  not  been  tested  further  than  Fx.  Since 
de5  and  delt  have  proven  to  be  identical,  the  crosses  24  and  38 
represent  the  same  combinations  as  65  and  89.  This  leaves  only 
7  distinct  combinations  which  have  not  yet  been  made.  There 
is,  however,  some  additional  evidence  in  several  of  the  untested 
combinations  which  indicates  that  the  defectives  involved  are  not 
alike.  Crosses  88  and  94,  representing  combinations  of  de±  by 
de12  and  de13  respectively,  will  certainly  give  di-hybrid  segregation 
in  F2  because  de4  is  a  "germless"  defective  while  de12  and  derj 
both  produce  embryos.  Crosses  75  and  90,  representing  com- 
binations of  de6  by  de12  and  de14  respectively,  should  also  give  de- 
hybrid  segregation  because  dee  is  linked  with  sugary,  as  recorded 
later,  while  de12  and  de14:  are  not.  The  same  is  true- of  the  cross 
100  between  dex  and  de12.  The  former  is  linked  with  sugary,  the 
latter  not.  This  leaves  only  two  combinations,  59  and  69,  about 
which  there  is  any  doubt. 

The  evidence,  then,  is  almost  conclusive  in  indicating  that  thirteen 
distinct  genetic  factors  for  defective  seeds  are  involved  in  the  four- 
teen stocks  tested.  There  is,  of  course,  the  possiblity  that  one  of 
the  two  doubtful  combinations  will  reveal  an  additional  case  of  two 
stocks  which  are  genetically  identical,  even  though  these  com- 
binations are  between  defectives  which  differ  decidedly  in  appear- 
ance. It  must  be  remembered  that  two  characters  may  be 
genetically  identical  in  one  main  factor  and  yet  differ  pheno- 
typically  because  of  minor  modifying  factors.  This  is  shown  by 
the  cross  between  de5  and  de1±  which  proved  these  two  defective's 
to  be  identical  although  they  were  far  apart  in  the  arbitrary  series 
which  had  been  arranged  on  the  basis  of  external  appearance. 


ENDOSPERM    CHARACTERS    IN    MAIZE  531 

These  results  give  some  indication  of  the  enormous  number  of 
■distinct  defective  seed  types  which  probably  occur  in  maize.  A 
sample  of  fourteen  types  was  taken  at  random  from  the  grab  bag 
which  constitutes  the  germplasm  of  maize,  and  thirteen  of  these 
proved  to  be  genetically  distinct.  The  total  number  of  distinct 
lethal  seed  factors  in  the  germplasm  of  this  species  can  only  be 
conjectured,  but  it  probably  equals  or  exceeds  the  number  of  dis- 
tinct varieties  of  maize  which  are  now  grown. 

THE  MORPHOLOGY  OF  DEFECTIVE  SEEDS 

In  order  to  determine  the  irregularities  in  development  which 
cause  one-fourth  of  the  seeds  on  a  segregating  ear  to  be  defective 
while  the  remainder  are  normal,  and  to  find,  if  possible,  constant 
differences  which  distinguish  some  of  the  types  from  others,  a 
histological  examination  of  all  fourteen  types,  in  various  stages 
of  development,  has  been  made. 

The  material  was  killed  and  fixed  in  Carnoy's  solution,  a  mix- 
ture of  three  parts  of  absolute  alcohol  to  one  of  glacial  acetic 
acid.  Two  other  fixing  agents,  Benda's  solution  and  a  concen- 
trated solution  of  picric  acid,  were  also  tried.  The  former  gave 
excellent  results,  but  was  discontinued  because  of  the  high  cost 
of  osmic  acid,  its  most  important  constituent.  The  picric  acid 
solution  proved  to  be  very  unsatisfactory  because  of  the  difficulty 
of  removing  all  traces  of  the  discoloration  from  such  large  sec- 
tions. All  material  was  imbedded  in  paraffin,  cut  in  sections  of 
ten  microns  and  stained  in  Delafield's  haematoxylin.  Some  of  the 
sections  were  also  stained  in  a  dilute  solution  of  iodine  and 
potassium  iodide  to  bring  out  possible  differences  in  the  starch 
grains. 

THE   CYTOLOGICAL   MECHANISM    OF   ENDOSPERM    FORMATION 

The  cytological  details  of  the  mechanism  leading  up  to  the 
formation  of  the  endosperm  in  maize  are  fairly  well  established. 
When  Nawaschin,  in  1898,  made  the  discovery  that  the  endosperm 
of  Lilium  is  the  product  of  a  sexual  fusion  entirely  apart  from 
that  which  gives  rise  to  the  embryo,  three  investigators,  DeVries 
(1899),  Correns  (1899),  and  Webber  (1900),  simultaneously  and 
independently  reached  the  conclusion  that  this  mechanism  was 
probably  responsible  for  the  phenomenon  of  xenia  in  maize, 
although  it  was  not  until  1901  that  Guignard  furnished  the  cyto- 
logical evidence  of  double  fertilization  in  this  species. 

More  recently  Weatherwax  (1919)  and  Miller  (1919)  have 
independently  repeated  Guignard's  researches  and  both  have  given 
detailed  descriptions  and  illustrations  of  the  entire  process  lead- 
ing up  to  fertilization.  In  most  respects  the  accounts  of  these 
two  writers  agree  very  well,  though  Miller  believed  that  all  four 


532  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    2JO, 

megaspores  functioned  while  Weather  wax  observed  the  disinte- 
gration of  three  megaspores,  with  only  one  persisting.  In  an 
earlier  paper,  however,  Weatherwax  (1917)  also  had  made  the 
observation  that  all  four  megaspores  functioned  and  his  dis- 
covery that  only  one  persisted  was  made  only  after  attention  was 
called  to  the  disagreement  between  his  earlier  cytological  observa- 
tion and  certain  well  established  facts  regarding  the  genetic 
behavior  of  the  endosperm. 

When  the  pollen  tube  enters  the  micropyle,  two  identical  sperm 
are  emptied  into  the  embryo  sac.  One  of  these  fuses  with  the 
egg  and  an  embryo  is  produced ;  the  other  fuses  with  one  of  the 
polar  nuclei  which  lie  close  together  in  the  embryo  sac.  Almost 
immediately  the  fusing  nuclei  are  joined  by  the  second  polar 
nucleus,  this  process  constituting  the  ''triple  fusion"  characteristic 
of  many  angiosperms.  It  is  of  importance  to  note,  in  connection 
with  the  possible  explanation  of  some  of  the  forms  of  non-hered- 
itary defectives  described  later,  that  both  Weatherwax  and  Miller, 
in  repeated  observations,  never  found  the  two  polar  nuclei  fusing 
before  fertilization  of  one  of  them  had  occurred. 

The  endosperm  of  angiosperms  is  unique  in  that  it  is  the  product 
of  a  fusion  in  which  the  two  parents  do  not  contribute  equally. 
Two  maternal  nuclei,  with  their  assortment  of  chromosomes  bear- 
ing the  hereditary  factors,  combine  with  one  male  nucleus.  The 
female  parent,  therefore,  contributes  two  sets  of  chromosomes  and 
a  double  dose  of  the  assortment  of  hereditary  factors  while  the 
pollen  parent  contributes  only  one  set  of  chromosomes  and  a  single 
dose  of  factors. 

This  peculiar  situation  enabled  Hayes  and  East  (191 5)  to 
demonstrate  the  fallacy  of  the  "presence  and  absence"  conception 
of  dominant  and  recessive  factors.  These  writers  found  that  in 
crosses  between  flint  and  flour  varieties  the  inheritance  was  always 
apparently  maternal,  a  double  dose  of  the  maternal  condition  being 
always  dominant  to  a  single  dose  of  the  alternative  condition. 
In  other  words,  two  "absences"  were  dominant  to  a  single 
"presence." 

DEVELOPMENT   AFTER    FERTILIZATION 

The  general  features  of  the  development  of  the  endosperm  and 
embryo  in  the  cereals  are  fairly  well  established.  Details  of 
development  which  distinguish  maize  from  other  grasses  are 
gradually  being  added  as  special  phases  are  investigated.  True 
(1893)  and  Poindexter  (1903)  studied  the  general  development 
of  the  caryopsis.  Reed  (1904)  has  investigated  the  secreting 
cells  of  the  scutellum  of  maize.  Sargent  and  Robertson  (1905) 
have  made  a  very  thorough  study  of  the  anatomy  of  the  scutellum. 
The  aleurone  layer  has  been  the  subject  of  cytological  studies 
especially   by   Liidtke    (1890),    Haberlandt    (1890)    and    Groom 


ENDOSPERM    CHARACTERS    IN    MAIZE  533 

(1893).  The  successive  stages  in  the  development  of  the  embryo 
have  been  described  and  figured  by  Weather  wax  (1920). 

The  general  development  of  the  caryopsis  in  maize  is  briefly 
as  follows :  The  endosperm  fusion  nucleus  begins  division  almost 
at  once  and  the  rapidly  growing  endosperm  soon'  fills  the  embryo 
sac.  The  embryo  nucleus  does  not  divide  immediately  after 
fusion  and  the  first  division  usually  does  not  occur  until  after  the 
nuclei  of  the  endosperm  number  20  or  more.  (Miller,  1919.) 
The  nucellus  soon  begins  to  disintegrate  and  is  partly  absorbed, 
the  remainder  being  compressed  into  a  thin  integument  between 
the  pericarp  and  endosperm. 

By  the  time  that  the  early  milk  stage  is  reached,  the  endosperm 
occupies  the  entire  space  within  the  pericarp  and  exerts  consider- 
able pressure.  (See  Plate  XXI,  Fig.  1.)  ,The  embryo  on  the 
other  hand  is  still  rather  rudimentary.  Fom  this  point  on,  the 
embryo  develops  more  rapidly  than  the  endosperm,  the  latter 
undergoing  only  slight  additional  increase  in  size  while  the  former 
grows  rapidly,  pushing  further  and  further  into  the  endosperm 
tissue. 

Small  starch  grains  are  found  in  the  outer  cells  of  the  endo- 
sperm in  the  early  milk  stage,  which  in  the  writer's  material 
occurred  at  about  15-20  days  after  pollination.  By  the  time  that 
the  late  milk  stage  is  reached  at  about  25  days  to  four  weeks  after 
pollination,  the  cells  in  the  upper  part  of  the  endosperm  are  com- 
pletely packed  with  starch  grains,  although  those  in  the  lower  part 
are  still  relatively  clear.  In  material  fixed  after  this  stage,  the 
contents  of  the  cells  drop  out  in  sectioning  and  in  most  cases  no 
histological  studies  of  further  changes  have  been  made. 

The  aleurone  layer  is  present  in  most  specimens  in  the  early 
milk  stage,  though  no  color  can  be  detected  in  this  layer  in  un- 
sectioned  material  at  this  period. 

DEVELOPMENT  OF  DEFECTIVE  SEEDS 

In  preliminary  experiments  of  1922,  seeds  were  fixed  at  inter- 
vals of  1,  2,  4,  7,  10,  20  and  30  days  after  pollination.  The 
•defective  seeds  could  not  be  distinguished  from  the  normal  seeds 
in  the  early  stages  and  it  was  necessary  to  examine  sections  from 
a  large  number  of  seeds  in  order  to  be  certain  that  defectives  were 
included.  In  1923  no  material  was  collected  until  the  normals 
and  defectives  on  segregating  ears  could  be  distinguished  from 
each  other.  This  point  was  usually  reached  in  the  blister  or  early 
milk  stage,  or  at  about  fifteen  to  twenty  days  after  pollination  in 
most  of  the  types.  Some  of  the  partial  defectives,  however,  could 
not  be  distinguished  from  normal  seeds  on  the  same  ears  until 
later. 

Defectives  and  normal  seeds  were  always  removed  in  pairs  for 
comparison  and  the  seeds  were  usually  taken  from  the  middle  of 


534  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279' 

the  ear  to  avoid  differences  due  to  unequal  development  at  butts 
and  tips  of  the  ears.  Before  dropping  the  seeds  into  the  fixing 
agent,  as  much  tissue  as  possible  on  either  side  of  the  embryo  was 
removed  to  permit  a  more  rapid  penetration  of  the  solution. 

REGULAR  DEVELOPMENT   IN   EARLY  STAGES 

The  inheritance  of  all  of  the  defective  seed  types  as  simple 
Mendelian  recessives  pointed  to  a  regular  functioning  of  the  fertil- 
ization mechanism  and  the  fusion  of  male  and  female  gametes. 
The  specimens  collected  in  the  early  stages  bore  out  this  assump- 
tion. All  of  the  types  of  which  the  early  stages  after  pollination 
were  studied  showed  the  normal  beginning  of  endosperm  and 
embryo  formation  and  no  marked  differences  between  normal  and 
defective  seeds  were  noted. 

In  specimens  fixed  after  the  blister  or  early  milk  stage,  how- 
ever, the  differences  between  normal  and  defective  seeds  were  very 
striking.  The  various  types  of  defectives  differed  from  each 
other,  however,  only  in  general  development  and,  with  the  excep- 
tion of  one  type,  no  specific  morphological  differences,  which 
always  distinguish  one  type  from  another,  have  been  found.  A 
general  account  of  the  development  of  these  various  types  of 
aborted  seeds  follows. 

THE   PERICARP 

No  matter  how  defective  the  endosperm  and  embryo  may  be, 
the  pericarp  usually  attains  a  normal  or  almost  normal  develop- 
ment. This  affords  a  striking  illustration  of  the  complete  inde- 
pendence of  these  two  tissues  which,  though  borne  on  the  same 
plant,  represent  distinct  sporophyte  generations.  The  pericarp  is 
maternal  in  its  origin  and  with  the  exception  of  the  stimulus  from 
pollination  which  sets  off  its  development,  it  does  not  appear  to 
be  influenced  by  the  hereditary  composition  of  the  new  sporophyte 
which  it  encloses. 

In  normal  seeds,  the  pericarp  is  constantly  distended  by  the  pres- 
sure of  the  growing  endosperm.  In  the  defectives,  there  is  always 
a  space  between  these  tissues.  In  early  stages  this  space  is  filled, 
partly  with  nucellar  tissue  and  partly  with  a  clear  watery  solution. 
In  later  stages  the  walls  of  the  ovule  are  pushed  together  by  the 
pressure  of  the  normal  seeds  on  either  side  and  the  space  dis- 
appears as  shown  in  Plate  XXI,  which  shows  three  successive 
stages  in  the  development  of  the  de14  type  of  defective  seeds. 

THE  NUCELLUS 

In  normal  seeds  the  nucellus  rapidly  disintegrates  following 
fertilization.  Part  of  it  is  probably  absorbed  while  the  remainder 
is  compressed  into  a  thin  integument  between  the  endosperm  and 


ENDOSPERM    CHARACTERS    IN    MAIZE  535 

pericarp  and  soon  loses  its  identity  as  a  separate  tissue.  (Plate 
XXL)  The  nucellus,  like  the  pericarp,  is  of  maternal  origin  and 
is  not  influenced  by  the  sporophyte  with  which  it  is  in  constant 
contact,  except  that  in  the  absence  of  a  vigorous  and  rapidly  grow- 
ing endosperm  it  is  permitted  to  persist  as  a  distinct  tissue  for 
longer  periods  than  it  does  in  normal  seeds. 

THE  ENDOSPERM 

The  endosperm  of  the  defective  seeds  differs  from  that  of 
normal  seeds  in  degree  rather  than  in  kind.  In  no  case  does  it 
attain  the  size  of  the  normal  endosperm,  but  in  details  of  develop- 
ment no  marked  differences  are  noted.  With  regard  to  the 
development  of  the  endosperm,  the  defectives  of  these  fourteen 
stocks  form  a  continuous  series  ranging  from  the  delz  and  dexi 


Fig.  55. — Three  successive  stages  of  development 
of  defective  seeds  of  dew.  No  aleurone  layer  is 
found  at  any  stage.  (Figures  55-58  represent  a 
magnification  of  approximately  6.5  diameters.) 


types  in  which  only  a  small  mass  fo  tissue  is  present,  to  the  dex 
and  de5  types,  in  which  the  endosperm  is  fully  half  size. 

With  regard  to  the  rate  at  which  the  endosperm  increases  in 
size  from  week  to  week  the  same  gradation  is  found.  In  the  de14: 
stock,  for  example,  the  defective  seeds  at  the  early  stage  have  only 
a  small  mass  of  endosperm  tissue.  At  the  late  milk  stage  no 
increase  in  size  is  noted,  and  at  the  early  dough  stage  the  develop- 
ment of  the  endosperm  still  remains  practically  the  same,  as 
shown  in  Plate  XXI.  In  partial  defectives,  such  as  those  of  the 
de5  stock,  for  example,  the  endosperm  gradually  increases  in  size 
from  week  to  week  though  its  rate  of  growth  is  considerably 
retarded  as  compared  to  the  endosperm  of  normal  seeds  and  its 


536 


CONNECTICUT    EXPERIMENT    STATION 


BULLETIN    2/9 


final  size  is  considerably  less  than  that  of  a  normal  endosperm. 
(Plate  XXII.) 

The  order  of  the  fourteen  stocks  of  defective  seeds  on  the  basis 
of  their  endosperm  development  as  shown  by  these  histological 
studies  is  not  exactly  the  same  as  the  arrangement  made  on  the 
basis  of  external  appearance  alone.  In  general,  however,  the  two 
series  agree  very  well. 

STARCH   GRAIN  FORMATION 

The  same  general  differences  noted  in  size  of  endosperm  are 
also  found  with  regard  to  the  formation  of  the  starch  grains.  In 
the  defectives  at  the  lower  end  of  the  series  no  starch  grains  were 
found  at  any  stage  examined.  Types  further  up  in  the  series 
produce  small  starch  grains  in  the  periphery  of  the  endosperm  in 
later  stages,  while  in  the  partial  defectives,  starch  grains  are  found 


Fig.  56. — Defective  seeds  of  den  at  early  milk 
stage ;    dei,  late  milk ;    den,  late  milk. 


in  every  stage  examined,  and  in  later  stages  the  cells  of  the  endo- 
sperm are  packed  with  starch  grains  which  are  apparently  normal 
in  size  and  structure. 

No  characteristic  differences  in  the  structure  of  starch  grains 
were  noted  with  the  exception  of  the  defective  seeds  of  de±,  In 
the  recessive  seeds  of  this  strain  the  starch  grains  have  the  appear- 
ance of  undergoing  hydrolosis  many  of  them  being  completely 
broken  up. 

THE  ALEURONE  LAYER 

The  defectives  at  the  foot  of  the  series  produce  no  aleurone 
layer  at  any  stage  examined  and  this  might  be  expected  since  an 
aleurone  layer  is  usually  formed  only  in  later  stages  of  normal 
development.      Beginning  with  delt,  however,  an  aleurone  layer 


ENDOSPERM    CHARACTERS    IN    MAIZE 


537 


is  found  in  the  later  stages  examined.  In  some  cases  this  layer 
extends  only  part  way  around  the  endosperm,  in  others  it  com- 
pletely surrounds  this  structure  except,  of  course,  at  the  base 
where  no  aleurone  layer  is  found  even  in  normal  seeds.  Draw- 
ings in  which  the  aleurone  layer  has  been  enlarged  out  of  propor- 
tion to  the  other  structures  are  shown  in  figures  55-58.  In 
defectives  higher  in  the  series  than  de10,  an  aleurone  layer  is  found 
in  almost  all  stages  examined. 


THE  EMBRYO 


With  the  exception  of  de±  all  of  the  types  were  found  to  con- 
tain embryos.  This  type  is  similar  to  the  "germless"  seeds 
reported  by  Demerec  (1923)  which  at  maturity  contain  no  embryo. 


Fig.  57. — Defective  seeds  ,of  den  de-3  and  des  at  approxi- 
mately the  same  stage,  showing  difference  in  degree  of  develop- 
ment of  embryo,  endosperm,  and  aleurone  layer. 


No  paraffin  sections  of  this  defective  were  secured  because  the 
recessive  seeds  are  distinguishable  from  the  normals  only  shortly 
before  maturity,  at  which  time  the  seeds  are  too  corneous  to  be 
sectioned  by  the  paraffin  method.  Free-hand  dissection  in  early 
stages  of  seeds  from  ears  later  proven  to  be  segregating  for  this 
character,  showed  that  an  embryo  was  present  in  all  the  seeds. 
Apparently  the  embryo  is  digested  and  absorbed  later.  The  cavity 
which  remains  on  the  germinal  side  of  the  seed  is  partly  filled  with 
a  hard  brittle  mass  of  substance  with  no  definite  structure.  As 
already  mentioned,  the  endosperm  of  this  type  also  shows  evidence 
of  digestion. 


538 


CONNECTICUT    EXPERIMENT    STATION 


BULLETIN    279 


In  general,  there  is  a  marked  correlation  between  the  develop- 
ment of  the  embryo  and  that  of  the  endosperm.  The  complete 
defectives  in  which  the  endosperm  remains  practically  stationary 
have  very  rudimentary,  undifferentiated  embryos  which  show  no 
further  development  after  the  early  milk  stage.  (Plate  XXL) 
Defectives  higher  in  the  series  show  some  increase  in  size  from 
week  to  week,  but  no  clear  differentiation  of  various  parts  of  the 
embryo  is  apparent  though  a  "growing  point"  is  indicated  by  a 
greater  concentration  of  nuclei  in  certain  regions.  Beginning 
with  des,  a  scutellum,  coleptile  and  several  rudimentary  leaves  are 
distinguished,  while  in  some  of  the  partial  defectives  the  develop- 
ment of  the  embryo  is  fairly  normal. 

The  embrvos  of  the  defective  seeds  often  show  considerable  dis- 


Fig.  58. — Three  successive  stages  of  den.  No  aleurone 
layer  is  found  in  the  early  stage  but  a  partial  or  com- 
plete layer  is  formed  later.  Note  the  distortion  of  the 
embryo  in  the  final  stage. 

tortion  in  shape.  In  later  stages  this  may  be  attributed  to  the 
pressure  exerted  by  the  normal  seeds  on  either  side  as  shown 
in  Plate  XXIII,  but  in  some  cases  a  distortion  is  noted  even  when 
the  defective  seeds  have  not  been  under  pressure.  The  embryo 
tends  to  be  short  and  "blocky"  as  shown  in  Plate  XXII,  which 
may  be  compared  to  Plate  XXIV  which  shows  the  successive 
stages  in  the  development  of  the  normal  embryo. 


GENERAL  ASPECTS 


Aside  from  the  interesting  demonstration  that  the  pericarp,  once 
its  development  is  stimulated  by  pollination,  proceeds  quite  inde- 
pendently of  the  tissues  which  it  encloses,  the  most  outstanding 


ENDOSPERM    CHARACTERS    IN    MAIZE  539 

feature  of  the  morphology  of  the  defective  seeds  is  the  marked 
correlation  between  the  development  of  the  endosperm  and  embryo. 

An  external  examination  of  some  of  the  defective  seeds  had 
led  to  the  assumption  that  these,  in  many  cases,  contained  no  tissue 
whatever.  It  was  surprising,  therefore,  to  find  distinct,  though 
rudimentary,  endosperm  and  embryo  at  some  stage  in  every  type 
examined.  It  was  even  more  surprising  to  find  that  the  delete- 
rious influence  of  the  lethal  factors,  which  they  carried,  affected 
both  of  these  structures  to  almost  the  same  degree.  It  might  be 
supposed  that  some  factors  would  affect  specifically  the  embryo, 
permitting  the  endosperm  to  continue  in  a  more  or  less  normal 
fashion.  Others  might  be  expected  to  inhibit  particularly  the 
endosperm.  This  has  not  been  found  to  be  the  case.  Even  in 
the  dei  type,  which  lacks  an  embryo  at  maturity,  the  development 
of  both  structures  proceeds  normally  in  the  early  stages  and  the 
influence  which  later  destroys  the  embryo  also  has  a  very  marked 
effect  upon  the  endosperm. 

Although  there  is  still  some  difference  of  opinion  among  morph- 
ologists  as  to  the  real  nature  of  the  endosperm  in  angiosperms, 
from  the  standpoint  of  the  geneticist  it  has  always  been  considered 
a  sporophyte,  differing  from  its  near  relative  the  embryo,  in  struc- 
ture, in  capacity  for  continued  development,  and  in  ability  to  repro- 
duce. In  the  expression  of  the  hereditary  factors  which  they 
receive,  the  endosperm  and  embryo  are  fundamentally  alike  and 
eventually  the  morphologists,  too,  may  come  to  regard  the  endo- 
sperm of  angiosperms  as  a  modified  sporophyte. 


Table  3.    Average  Weight  of  Seeds  from  Ears  Harvested  at  Successive 
Stages  of  Maturity. 


Days  after 

r 

— Av.  wt. 

in  mg- 

"V 

Relative 

Pollination 

1 

2 

3 

Av. 

Development 

14 

5 

5 

5 

5 

1-5 

21 

25 

27 

37 

30 

9.1 

28 

82 

86 

71 

80 

24.2 

35 

in 

97 

126 

III 

33-6 

41 

159 

166 

189 

171 

51.8 

51 

234 

222 

226 

227 

68.8 

75 

374 

319 

306 

330 

1 00.0 

PHYSIOLOGY  OF  DEFECTIVE   SEEDS 

Histological  examinations  of  the  normal  and  defective  seeds 
were  confined  largely  to  a  rather  brief  period  beginning  with  the 
blister  stage  and  ending  when  the  seeds  had  reached  the  dough 
stage.  In  order  to  determine  the  differences  between  normal  and 
defective  seeds  in  later  stages  of  development,  a  comparison  of 
their  rate  of  growth,  final  dry  weight,  percentage  of  germination 


54°  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    2/0, 

and  effect  upon  the  sporophyte  and  gametophyte  in  the  haploid 
condition  was  made. 


BATE   OF   GROWTH,    FINAL   WEIGHT  AND   GERMINATION   OF 
NORMAL  SEEDS 

The  rate  of  growth  of  normal  seeds  of  maize  was  determined 
as  follows :  A  large  number  of  plants  of  an  F1  hybrid  of  homozy- 
gous inbred  strains  were  grown  under  uniform  soil  conditions. 
These  plants,  all  being  genetically  alike,  came  into  silk  and  were 
pollinated  at  about  the  same  time.  At  two  weeks  after  pollina- 
tion, and  at  intervals  thereafter  until  maturity,  three  ears  were 
taken  at  random  from  these  plants.  The  ears  were  reduced  to 
an  air  dry  condition  after  which  the  kernels  were  removed, 
counted,  weighed  "en  masse"  and  the  average  weight  at  each  stage 
determined  by  simple  division. 

It  is  quite  possible  that  in  ears  harvested  at  various  stages  of 
maturity  in  this  way  there  is  a  transfer  of  materials  from  the  cob 
to  the  kernels  during  the  drying  out  process.  Such  an  exchange, 
if  it  occurs  at  all,  would  probably  be  proportionate  for  the  various 
stages  and  is  not  regarded  as  a  serious  source  of  error. 

Table  4.     Percentage  Germination  of  Corn  Seeds  Harvested  at  Successive 
Stages  of  Maturity. 
Days  After  Pollination     Relative  Development        Percent  Germination 

14  1-5  O 

21  9.1  28 

28  24.2  56 

35  33-6  72 

41  51.8  92 

51  68.8  96 

75  1 00.0  98 

The  average  weights  in  milligrams  and  the  relative  weights,  as 
compared  to  the  final  weight  at  maturity,  of  these  seeds  harvested 
at  various  stages  of  development,  are  given  in  Table  3.  Figures  1 
and  2  of  Plate  XXV  show  respectively  a  representative  ear  at  each 
stage  and  50  seeds  from  each  ear. 

The  50  seeds  representing  each  stage  of  development  were 
planted  in  sand  in  the  greenhouse  with  the  results  shown  by  the 
photograph  in  Plate  XXV,  Figure  3.  Some  germination  occurred 
in  every  lot  except  the  first  which  was  harvested  at  fourteen  days 
after  pollination.  The  percentage  of  germination  of  each  lot  and 
the  relative  weight  of  the  seeds  from  which  it  was  grown  are 
given  in  Table  4. 

The  normal  seeds  of  maize,  like  those  of  barley  (Harlan  and 
Pope,  1922),  are  apparently  capable  of  some  germination  at  all 
stages  of  development  but  the  very  earliest. 


ENDOSPERM    CHARACTERS    IN    MAIZE 


541 


RATE  OF  GROWTH,   FINAL  WEIGHT,  AND  GERMINATION   OF 
DEFECTIVE  SEEDS 

The  rate  of  growth  of  defective  seeds  as  compared  to  normal 
seeds  from  the  same  ears  was  determined  for  one  type,  de10. 
Ears  from  ah  Fx  hybrid  of  this  stock  crossed  with  an  unrelated 
inbred  strain  were  harvested  when  the  segregation  on  the  ears  first 
became  apparent  and  at  intervals  thereafter  until  maturity.  When 
all  the  ears  had  been  reduced  to  dryness,  the  kernels  were  shelled 
off,  the  normal  and  defective  seeds  separated,  counted  and 
weighed,  and  the  average  weight  of  each  class  determined.  The 
growth  curves  of  the  normal  and  defective  seeds  from  ears  segre- 
gating for  de10  are  shown  in  Figure  59.     Although  the  rate  of 


300 


200 


100 


» 


De 


de 


20        30        40 
DAYS  AFTER  POLLINATION 


50 


60 


Fig.  59. — Growth  curves  of  normal  and  defective  seeds  from 
the  same  segregating  ears  of  the  dew  stock. 

increase  in  dry  weight  is  very  low  for  the  defective  seeds,  the 
two  curves  appear  to  be  of  the  same  general  type. 

In  determining  the  relative  development  at  maturity  of  the  four- 
teen types,  dry  weight  was  used  as  a  criterion.  Obviously,  the 
relative  development  cannot  be  determined  with  any  high  degree 
of  accuracy  because  it  is  influenced  to  some  extent  by  the  environ- 
ment and  considerably  by  the  hereditary  constitution  of  the  stocks, 
certain  of  the  types  showing  more  development  in  crosses  than  in 
inbred  strains.  In  order  to  make  the  determinations  as  nearly 
comparable  as  possible,  the  figures  on  relative  development  have, 
with  one  exception,  been  taken  from  crosses  which  were  all  iden- 
tical with  respect  to  one  of  the  parents,  and  all  grown  the  same 
season.  In  this  way  the  defectives  are  compared  under  condi- 
tions in  which  hereditary  and  environmental  differences  are 
reduced  to  a  minimum. 


542  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

The  relative  development  of  defective  seeds  at  maturity  of  each 
of  the  fourteen  stocks  is  shown  in  Table  5. 

Table  5.    Relative  Development  in  Defective  Seeds  of  Fourteen  Stocks  as 
Compared  to  Normal  Seeds  on  Same  Ears.  • 


Stock 

No.  Ears 

Relative  Devel 

dei 

6 

504 

des 

3 

58.8 

de3 

3 

29-9 

dei 

3 

37-3 

des 

2 

18.0 

des 

3 

34-0 

der 

3 

18.8 

des 

3 

15.0 

dea 

3 

5-9 

deio 

2 

13.7 

den 

3 

7-3 

dei2 

3 

40 

den 

2 

4-5 

den 

3 

2.4 

The  types  show  a  range  in  development  from  2.4  per  cent  in 
the  de14:  type  to  58.8  in  the  de2  type.  It  is  noted  that  the  order 
of  these  types  based  on  their  relative  development  is  not  the  same 
as  that  arranged  on  the  basis  of  external  appearance  alone.  This 
is  partly  due  to  the  fact  that  the  original  arrangement  was  based 
on  inbred  strains  while  the  relative  development  has  been  deter- 
mined from  hybrids.  Even  when  all  are  crossed  with  the  same 
unrelated  stock,  some  of  the  types,  particularly  2,  7,  and  10,  show 
a  greatly  increased  development  after  crossing.  In  some  crosses 
the  de2  type  reappears  in  the  second  generation  so  altered  in 
appearance  that  it  is  scarcely  recognized.  The  recessive  seeds  are 
almost  equal  to  normal  seeds  in  size  and  weight,  and  differ  from 
the  latter  only  in  a  paler  color  and  a  mottled  appearance. 

Whether  this  increase  in  development  which  follows  crossing  is 
due  to  the  greater  vigor  of  the  plants  on  which  they  are  borne, 
or  to  the  action  of  modifying  factors  contributed  by  one  or  both 
parents,  is  not  known.  That  some  of  the  defectives  are  influ- 
enced by  modifying  factors,  is  almost  certain.  The  de5  and 
delt  types,  for  example,  are  genetically  alike,  yet"  differed  in 
appearance,  not  only  in  the  original  stocks,  but  in  hybrids  in  which 
both  were  crossed  to  the  same  unrelated  stocks. 

Judging  from  the  dry  weight  alone,  the  fourteen  types  of 
defectives  correspond  to  various  stages  in  the  development  of  the 
normal  seed.  This  is  shown  diagramatically  in  Figure  73  (Part 
III)  in  which  the  relative  development  of  each  type  is  represented 
by  a  point  on  the  normal  growth  curve  of  maize  seeds. 

At  first  glance,  the  defective  seeds  are  comparable  to  normal 
seeds  which  have  had  their  development  arrested  at  an  early  stage, 


ENDOSPERM    CHARACTERS    IN    MAIZE 


543 


as  has  occurred  in  the  ears  harvested  at  successive  stages  of 
development,  shown  in  Plate  XXV.  When  these  various  defec- 
tive seeds  are  tested  for  germination,  however,  it  is  found  that  they 
are  by  no  means  equal  to  normal  seeds  of  the  same  relative 
development.  This  is  shown  by  the  figures  in  Table  6,  in  which 
the  germination  of  each  type  of  defective  is  compared  to  the 
theoretical  germination  of  immature  normal  seeds  of  the  same 
relative  development.  The  theoretical  germination  of  the  normal 
seeds  was  determined  by  interpolation  of  the  figures  in  Table  4. 

It  is  noted  that  the  defective  seeds  in  every  case  show  a  much 
lower  germination  than  would  be  expected  from  normal  seeds  of 


Fig.  60. — Seedlings  from  normal  and  defective  seeds  of  dez.  Only 
rarely  do  the  seedlings  from  the  recessive  seeds  survive  more  than 
a  few  weeks. 


similar  development.  In  fact,  the  types  below  de7  showed  no 
germination  whatever  in  this  test  with  the  exception  of  one  seed 
from  a  total  of  131  seeds  of  the  delx  type.  Defective  seeds  of 
des  have  also  shown  slight  germination  in  some  cases,  but  in  the 
ears  used  in  this  particular  test  no  germination  occurred. 

Not  only  are  the  defective  seeds  less  able  to  germinate .  than 
normal  seeds  of  the  same  development,  but  those  which  do  succeed 
in  sprouting  produce  very  weak  seedlings  which  are  lacking  in 
vigor  and  soon  die.  Seedlings  from  normal  and  defective  seeds 
of  the  de2  stocks  are  shown  in  Figure  60.  By  planting  large 
numbers  of  seeds  in  the  greenhouse  and  transplanting  the  most 


544  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

vigorous  of  the  seedlings  to  the  field,  it  has  been  possible  to  obtain 
homozygous  plants  of  stocks  i,  2,  3  and  6. 

The  behavior  of  the  plants  grown  from  homozygous  defectives 
is  in  striking  contrast  to  those  which  result  when  immature  normal 
seed  is  grown.  In  appearance  the  two  lots  of  seed  are  almost 
identical,  both  being  badly  shrivelled  and  aborted.  Both  types 
give  a  low  germination  and  the  seedlings  are,  in  both  cases,  very 
weak  and  spindling.  The  plants  which  are  genetically  normal, 
however,  soon  recover  from  the  handicap  of  the  poor  food  supply 
which  the  aborted  seed  affords,  and  by  flowering  time  are  fully 
normal  in  stature,  and  yield  practically  as  much  grain  as  plants 
which  grow  from  well  filled,  mature  seed.  The  homozygous 
defectives,  on  the  other  hand,  remain  very  weak  and  spindling 
throughout  the  season,  produce  only  a  small  amount  of  pollen, 
and  rarely  any  ears.  At  least  one  pure  defective  ear  has  been 
obtained,  however,  from  each  of  the  four  stocks  mentioned  above, 
during  the  past  five  seasons.  Figure  61  shows  a  normal  and  pure 
defective  ear  from  the  de3  stock. 


Table  6.    The  Percentage  of  Germination  in  Defective  Seeds  of  Fourteen 

Stocks  Compared  to  the  Theoretical  Germination  of  Normal 

Seeds  of  the  Same  Relative  Development. 


Stock 

Defectives 

Theoretical  Normal 

d& 

45-6 

91.0 

de* 

44.9 

94-0 

de& 

54-0 

66.5 

de* 

3-5 

75-5 

de& 

1 1.2 

47-5 

dee 

11.8 

72.5 

dd 

19.6 

49.0 

de& 

0 

42.0 

det 

0 

17.0 

deio 

0 

39-0 

den 

0.8 

22.0 

dei2 

0 

10.0 

de-a 

0 

12.5 

den 

0 

3-5 

The  fact  that  the  germination  of  the  defective  seeds  is  con- 
siderably lower  than  that  of  immature  normal  seeds  of  the  same 
relative  development,  that  the  seedlings  of  all  types  are  extremely 
weak,  and  that  even  the  most  vigorous  ones  which  are  able  to 
survive  make  a  very  feeble  growth  throughout  the  season,  indi- 
cates that  these  lethal  and  semi-lethal  factors  do  more  than  merely 
arrest  the  seeds  at  a  certain  stage;  apparently  these  factors  in  a 
homozygous  condition  have  a  deleterious  influence  on  the  sporo- 
phyte  at  any  stage  in  which  they  are  given  an  opportunity  for 
expression.     In  most  of  the  types  the  deleterious  influence  is  so 


ENDOSPERM    CHARACTERS    IN    MAIZE 


545 


marked  that  the  career' of  the  new  sporophyte  is  brought  to  an 
end  in  the  seed  stage  and  the  lethal  factors  have  no  opportunity 
for  further  damage  past  this  period. 


Fig.  6i. — Left;  ear  segregating  de3.  Center;  self-pollinated  ear 
from  a  plant  homozygous  for  de3.  Right ;  open-pollinated  ear  from 
homozygous  plant. 


THE  INFLUENCE  OF  LETHAL  FACTORS  IN   HETEROZYGOUS  CONDITION 

Since  the  effect  of  the  lethals  is  so  marked  when  they  are  in 
homozygous  condition,  it  might  be  questioned  whether  they  have 


546  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    2/9 

not  a  similar  unfavorable  influence,  though  in  a  smaller  degree,  in 
the  heterozygous  condition. 

The  only  evidence  bearing  on  this  question  is  that  secured  from 
a  comparison  of  the  height  of  segregating  and  non-segregating 
plants  among  the  1089  self -pollinated  F1  plants  which  were  grown 
in  1923  and  1924.  In  1923  the  height  to  base  of  tassel  on  398  F1 
plants  was  measured.  The  average  heights  of  the  plants  which 
later  proved  to  be  free  of  these  lethal  factors  was  77.9  inches  as 
compared  to  77.2  for  those  which  segregated  for  a  single  type  of 
defective  and  73.7  for  those  which  segregated  for  two  types.  In 
1924,  659  Fx  plants  were  measured.  The  non-segregating  plants 
averaged  73.9  inches  in  height  as  compared  to  71.9  for  the  other 
two  groups.  Though  the  difference  in  height  is  not  significant 
either  year,  the  fact  that  the  two  groups  of  segregating  plants  are 
somewhat  lower  both  years  than  the  non-segregating  plants  may 
be  of  some  significance.  Table  7  gives  the  frequency  distribution 
of  the  segregating  and  non-segregating  plants  for  these  two  years. 

Table  7.     Frequency  Distribution  in  Height  of  Fi  Plants  from  Crosses  of 
Defective  Seed  Stocks. 

, Mid-Class  Values  in  Inches s 


Type  of  Plant  42  47      52  57  62  67  72     77  82  87  92  97  102  Total  Mean 

Segregating  3:1  3  7      9  22  51  72  86  117  98  52  12  2  ..  531    .  73.85  ±  .20 

Segregating  9:7  ..  ..        1  6  9  15  23     20  17  9 100  73.30  ±  .5; 

Total  segregating 

plants  3  7  10  28  60  87  109  137  115  61  12  2  ..  631  73.80  ±.3f 

Not  segregating  ..  5      8  21  29  45  78    89  69  54  15  2  1  416  74.90  ±  .3? 

Difference  in  favor  of  non-segregating  plants  i.io±  4<. 

Although  no  data  on  the  yield  of  segregating  and  non-segre- 
gating plants  can  be  secured  because  the  ears  are  artificially  polli- 
nated and  full  ears  are  seldom  obtained,  it  has  often  been  noted 
that  the  segregating  ears  have  a  tendency  to  be  smaller  than  those 
which  are  uniformly  normal. 

These  results  and  observations  cannot  be  regarded  as  more  than 
mere  indications  that  the  lethals  do  have  an  effect  in  the  heterozy- 
gous condition ;  they  certainly  do  not  afford  conclusive  evidence 
in  favor  of  such  an  assumption.  The  effect,  if  any,  of  the  lethals 
when  in  combination  with  their  dominant  allelomorphs  is  so 
slight  that  it  could  be  accurately  detected  only  by  a  delicate  test. 
Such  a  test  is  now  being  made  with  the  inbred  strains  which  have 
mutated  to  defective  tseeds,  and  which  are  presumably  homozygous 
for  all  factors  with  the  exception  of  a  single  pair  involving  the 
defective  factor  and  its  dominant  allelomorph. 

EFFECT  UPON  THE  GAMETOPHYTE 

The  gametophyte  generation  is  ordinarily  assumed  to  be  inde- 
pendent of  the  influences  of  the  genetic  factors  distributed  on  the 
chromosomes  which  it  carries.      Recent  evidence    (Jones,   1924^1 


ENDOSPERM    CHARACTERS    IN    MAIZE 


547 


(Mangelsdorf  and  Jones,  1926)  indicates,  however,  that  there  are 
exceptions  to  the  rule  and  that  in  some  cases,  the  gametophytes 
from  the  same  plant  are  not  all  alike  in  their  ability  to  reach  the 
micropyle  and  accomplish  fertilization.  This  brings  up  the  ques- 
tion of  the  effect  of  the  lethal  factors  on  the  gametophyte  genera- 
tion. Since  these  factors  have  such  a  marked  deleterious  influence 
on  the  sporophyte  at  all  stages,  might  they  not  have  some  degree 
of  expression  in  the  gametophyte  generation  as  weil  ? 

It  has  already  been  noted  that  there  is  a  deficiency  of  recessive 
seeds  on  segregating  ears  of  a  number  of  these  types  and  such  a 
condition  might  be  explained  by  slower  rate  of  growth  of  the 
pollen  tubes  carrying  the  lethal  factors. 

In  order  to  answer  this  question,  a  large  number  of  segregating 
ears  of  the  de3  stock  which  regularly  shows  a  deficiency  of  the 
recessives  were  divided  arbitrarily  into  top  and  bottom  halves 
and  the  normal  and  defective  seeds  in  each  half  were  counted 
separately.  If  there  were  a  constant  difference  in  the  rate  of 
growth  between  pollen  tubes  carrying  the  lethal  factors  and  those 
carrying  the  dominant  allelomorphs,  then  the  greater  difference 
which  the  pollen  tubes  were  forced  to  travel  in  reaching  the  ovules 
at  the  base  of  the  ears  would  act  against  those  carrying  the  lethal 
factors  and  cause  a  greater  deficiency  of  the  defective  seeds  in  the 
lower  halves  of  the  ears. 

The  results  of  making  counts  in  the  upper  and  lower  half  of 
fifteen  ears  segregating  for  de3  are  shown  in  Table  8. 


Table  8.     Normal  and  Defective   Seeds   on  Top  and   Bottom   Halves   of 
Ears  Segregating  dez. 


1 Top 

Half , 

, —  Bottom 

Half  -, 

,— -Percent  D 

efective- 

Ear  No. 

Normal 

Defective 

Normal 

Defective 

Top 

Bottom 

IO 

I65 

60 

204 

50 

26.7 

197 

29 

I46 

48 

158 

48 

24.7 

23-3 

30 

154 

44 

145 

39 

22.2 

21.2 

33 

89 

24 

78 

36 

21.2 

31-6 

45 

IOX) 

57 

207 

54 

23.I 

20.7 

46 

210 

63 

200 

65 

23.1 

24-5 

5i 

156 

44 

178 

36 

22.0 

16.8 

55 

155 

45 

185 

55 

22.5 

22.9 

56 

138 

40 

172 

60 

22.5 

25.9 

58 

145 

56 

171 

50 

27.9 

22.6 

120 

119 

32 

144 

48 

21.2 

25.0 

135 

119 

32 

79 

3i 

21.2 

28.2 

139 

145 

47 

96 

3i 

24.5 

24.4 

148 

125 

4i 

145 

42 

24.7 

22.5 

158 

115 

46 

112 

41 

28.6 

26.8 

Total 

2171 

679 

2274 

686 

23.8 

23.2 

Ex  3:1 

2137-5 

712.5 

2220 

740 

Deviations 

33-5 

46 

P.  E. 

15.6 

15-9 

Dev./P.  E. 

2.2 

2.9 

54§  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    2/9 

Although  the  percentage  of  recessives  in  the  bottom  halves  of 
these  ears  is  lower  than  that  in  the  top  halves,  the  difference  is 
not  significant.  When  the  ears  are  examined  individually  it  is 
noted  that  the  percentage  of  recessives  is  lower  in  the  top  half 
almost  as  frequently  as  in  the  bottom  half. 

From  these  results  it  may  be  concluded  that  the  lethal  factor? 
have  very  little,  if  any,  effect  upon  the  rate  of  pollen  tube  growth. 
Even  very  slight  differences  in  the  ability  of  the  pollen  tubes  to 
reach  the  micropyle  would  cause  marked  distortions  in  the  ratios 
and  should  result  in  different  proportions  of  defective  seeds  in 
the  upper  and  lower  halves  of  the  inflorescences. 

LINKAGE  RELATIONS 

Although  no  special  study  of  the  linkage  relations  of  these  char- 
acters has  yet  been  made,  it  was  to  be  expected  that  some  indica- 
tions of  linkage  would  be  encountered  as  by-products  of  the  other 
investigations.     This  proved  to  be  the  case. 

The  first  linkage  to  appear  was  that  between  de2  and  a  factor 
for  albino  seedlings.  This  linkage  has  been  briefly  mentioned  in 
a  previous  paper.      (Mangelsdorf,  1922.) 

Table  9.     Seedlings  from  Normal  and  Defective  Seeds  Showing  Linkage 
Between  de-i  and  w. 


r 

-Norrr 

al 

\ 

<_ 

Defect 

ve  N 

r  No. 

Green 

White 

Green 

White 

2 

126 

15 

30 

20 

4 

158 

19 

23 

19 

5 

79 

19 

O 

19 

7 

130 

20 

I 

39 

8 

119 

I 

28 

32 

Total  612  74  82  129 

As  already  noted,  the  de2  character  is  peculiar  in  that  it  is  greatly 
modified  in  certain  crosses.  In  a  cross  between  the  de1  and  de.2 
stocks  the  recessive  seeds  of  the  latter  reappeared,  so  altered  in 
appearance,  and  so  well  developed,  that  it  was  considered  feasible 
to  plant  a  row  of  them  in  the  field  in  order  to  secure  a  stock 
homozygous  for  this  factor.  A  week  later  when  the  seedlings 
had  emerged,  this  row  was  easily  the  most  conspicuovis  one  in  the 
field.  With  one  exception  all  of  the  seedlings  were  albinos,  and 
this  solid  row  of  pure  white  seedlings  in  striking  contrast  to  the 
normal  green  plants  on  either  side  furnished  a  most  striking 
demonstration  of  linkage. 

The  remaining  ears  of  this  cross  were  tested  in  the  greenhouse 
and  the  results  are  given  in  Table  9.  Figure  62  shows  the  seed- 
lings grown  from  the  normal  and  defective  seeds  from  one  of 
these  ears. 


ENDOSPERM    CHARACTERS    IN    MAIZE  549 

The  amount  of  crossing  over  between  de2  aire,  w  is  18  per  cent 
as  determined  from  the  normal  seeds  and  21.5  per  cent  as  deter- 
mined from  the  recessive  seeds. 

These  values  are  believed  to  be  somewhat  high  because  of  pos- 
sible inaccuracies  in  the  classification  of  normal  and  defective 
seeds  on  these  ears.  A  large  number  of  F3  progenies  were  there- 
fore grown.  In  these  the  development  of  the  defective  seed  was 
reduced,  and  the  segregation  so  well  defined  that,  it  is  believed, 


Fig.  62. — Seedlings  from  normal  and  defective  seeds  of  dei  show- 
ing linkage  between  defective  seeds  and  a  factor  for  white  seedlings. 


a  fairly  accurate  separation  was  made.  The  results  of  planting 
the  normal  and  defective  seeds  from  17  F3  ears  are  given  in 
Table  10. 

The  amount  of  crossing  over  as  determined  from  the  normal 
seeds  is  11.7;   from  the  defective  seeds,  10.5. 

As  nearly  as  can  be  determined  from  the  records  the  de2  stock 
is  the  same  one  in  which  the  w2  factor  for  white  seedlings  was 
found.  (Lindstrom,  1924.)  The  w2  factor  is  known  to  belong 
to  the  second  linkage  group  in  maize  and  it  is  probable,  there- 
fore, that  the  de2  factor  is  also  a  member  of  this  group  although 
further  tests  are  necessary  to  substantiate  such  an  assumption. 
Some  additional  evidence  for  it  exists  in  the  fact  that  Lindstrom 
(1923)  has  also  found  a  case  of  close  linkage  between  w2  and  a 
defective  seed  which  answers  the  description  of  the  de0  type. 


55°  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

LINKAGE  BETWEEN  SU  AND  de  FACTORS 

The  relation  between  the  factor  for  sugary  endosperm,  a  repre- 
sentative of  the  third  linkage  group,  and  the  fourteen  factors  for 
defective  seeds,  may  be  determined  from  the  crosses  between  de. 
and  the  thirteen  remaining  types.  The  de7  stock,  originally 
starchy,  had  been  changed  over  to  sugary  before  any  crosses 
were  made. 


Table  10.     Seedlings  from  Normal  and  Defective  Seeds  of  F3  Progenies 

Showing  Linkage  Between  de»  and  w. 

-Normal .  , Defective- 


Green  White  Green  White 

84  3  5  28 

89  5  16  22 

79  17  o  12 

83  9  2  32 
91  1  27  32 
54  7  1  16 

58  2  1  2 
42                         o                      o  6 

8302 

63  3                       1                      25 

21  232 

84  8  1  26 
81  12  12  33 
51  004 
74  9  1  11 
68  4                      o                      15 

59  2  2  15 

1089  87  72  283 

Since  the  defective  seeds,  with  the  exception  of  two  or  three 
types,  cannot  be  accurately  classified  with  regard  to  their  endo- 
sperm texture,  their  linkage  relations  with  sugary  must  be 
determined  from  the  normal  seeds  alone.  Linkage  of  sugary 
endosperm  with  any  of  the  lethal  factors  would  be  indicated  by  a 
distortion  of  the  normal  3  starchy :  1  sugary  ratio.  An  excess  of 
sugary  seeds  is  expected  when  the  sugary  and  defective  seed 
factors  enter  the  cross  from  opposite  parents  and  the  recessive 
factor  of  one  is  linked  with  the  dominant  allelomorph  of  the  other. 
With  complete  linkage  between  Sn  and  de,  2>ZV3  Per  cent  sugary 
seeds  in  the  normal  class  are  expected.  Thus  the  range  in  cross- 
ing over  from  50  per  cent  to  o,  corresponds  to  a  range  in  per- 
centage of  sugary  seeds  of  25  to  ZZTA-  This  is  shown  diagram- 
matically  in  Figure  63. 

F2  progenies  of  crosses  between  sugary  endosperm  and  all  of 
the  defective  seed  types  have  been  grown.  The  results  of  sepa- 
rating and  counting  the  starchy  and  sugary  seeds  in  the  normal 
class,  on  ears  from  all  of  these  crosses,  are  shown  in  Table  11. 


ENDOSPERM    CHARACTERS    IN    MAIZE 


551 


PERCENTAGE  OF  CROSSING-OVER 


Fig.  63. — Diagram  showing  the  theoretical  distortion  of  the  starchy : 
sugary  ratio  among  the  normal  seeds  when  a  defective  seed  factor  is 
linked  with  the  normal  allelomorph  of  sugary.  With  complete  linkage 
ZZVz  per  cent  sugary  seeds  are  expected  in  the  normal  class. 


55 2  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

Table  ii.     Segregation  of  Starchy  and  Sugary  among  the  Normal  Seeds 
of  Ears  also  Segregating  for  Defectives. 

Cross                     Ear  No.  Starchy  Sugary             %   Sugary 

deixsu                   947  71         36 

95i  213         89 

953  219         81 

956  98         32 

Total       601        238        28.4 
dei  x  su 


1024 

217 

91 

1034 

131 

42 

1035 

173 

52 

Total       521        185        26.2 


de3  x  su  1013        255 

1016  352 

1017  223 


Total       830        287        25.7 


dd  x  su                  1045  185  75 

1047  150  37 

1049  244  87 

Total  579  199        25.6 

de5  x  su                  1056  334  97 

1058  330  115 

1068  421  128 

Total  1085  340        23.0 

den  xsu                  1 107  66  37 

1 108  138  55 

Total  204  92        3 1. 1 

de-txsu                  1072  187  58 

1073  291  114 

1084  252  90 

Total  730  262        26.4 

deixsu                 2432  287  in 

2433  252  75 

Total  539  186        25.7 

dCn   X  SU                              IO96  200  62 

1098  177  50 

"             1 1 02  242  72 

Total  619  184        22.9 


ENDOSPERM    CHARACTERS    IN    MAIZE  553 

Table  II  (cont'd).     Segregation  of  Starchy  and  Sugary  among  the  Normal 
Seeds  of  Ears  also  Segregating  for  Defectives. 


Cross 

Ear  No. 

Starchy 

Sugary 

%   Sugary 

dew  x  su 

996 

278 

88 

24.O 

den  x  su 

969 
970 

971 

295 
137 
22$ 

104 
43 

54 

Total 

660 

201 

23-3 

dei2  x  su 

937 
938 
940 

306 
298 
170 

95 

105 

45 

Total 

774 

245 

24.0 

devi  x  su 

1075 
1079 

222 
135 

70 
5i 

Total 

357 

121 

25.3 

den  x  su 

« 

981 
988 
992 

162 

345 
231 

55 

in 

83 

Total 

738 

249 

25.2 

With  the  exception  of  the  cross  involving  de7,  the  lethal  factors 
and  the  factor  for  sugary  endosperm  have  been  contributed  by 
opposite  parents  and  linkage  would  be  indicated  by  an  excess  of 
sugary  seeds.  In  the  cross  of  de,,  the  de  and  su  factors  were 
introduced  by  the  same  parent  and  linkage  would  be  indicated  by 
a  deficiency  of  sugary  seeds.  . 

The  only  crosses  in  which  there  is  a  noticeable  distortion  ot 
the  starchy-sugary  ratio  are  those  involving  the  dex  and  de6 
factors.  In  the  former  28.4  per  cent  of  the  normal  seeds  are 
sugary.  This  is  an  excess  of  sugary  seeds  of  3.4  times  the  prob- 
able error  and  indicates  linkage  with  crossing  over  of  38.5  per 
cent.  Fortunately  the  defective  seeds  of  this  type  cap  also  be 
readily  classified  as  to  their  endosperm  texture  and  this  is  done 
in  Table  12.  _  ■  . 

Among  the  defective  seeds  there  is  a  marked  deficiency  ot 
sugary  individuals,  only  20.6  per  cent  being  found  as  compared  to 
28.4  per  cent  in  the  normal  class.  The  amount  of  crossing  over 
as  determined  from  the  defective  seeds  is  39  per  cent.  This 
agrees  very  closely  with  the  percentage  as  determined  from  the 
normal  seeds.  The  evidence  is  fairly  good,  therefore,  that  the 
de1  and  su  factors  are  linked. 


554  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

Table  12.     Segregation  of  Starchy  and  Sugary  among  the  Defective  Seeds 
from  Ears  of  a  Cross  dei  x  sur 

Ear  No.  Starchy 

947  42 

95i  75 

953  85 


Sugary 

%    Sugary 

10 
27 
18 

9 

19.2 

26.5 

17-5 
16.7 

64 
78 

20.9 
25.0 

956  45 

Total  247 

Ex.  3:1  233 

Deviation  14  — 14  4.4 

Probable  Error:  5.15 


In  the  cross  of  deQ  x  su,  31.1  per  cent  of  the  normal  seeds  were 
sugary.  This  is  an  excess  of  3.6  times  the  probable  error  and 
indicates  linkage  with  crossing  over  of  26  per  cent.  Unfortu- 
nately the  defectives  on  these  ears  could  not  be  classified  with 
regard  to  their  endosperm  texture  and  it  is  not  so  certain  that 
linkage  between  de6  and  su  exists,  although  it  is  strongly  indicated. 
An  excess  of  sugary  seeds  of  3.6  times  the  probable  error  would 
be  expected  as  a  chance  deviation  only  once  in  about  65  trials. 

The  cross  between  the  dex  and  de6  factors  shows  independent 
inheritance  of  these  two  characters  as  is  shown  in  Table  13.  This 
would  be  expected  even  though  both  are  linked  with  the  su  factor 
providing  that  the  loci  of  the  two  lethals  were  on  opposite  sides 
of  the  su  locus. 

Table   13.     Segregation   in   F2  of   a   Cross   between  dd   and  de&   Showing 
Independent  Inheritance. 


Ear  No. 

Normal 

Defective 

166 
168 

175 
178 

124 

117 
l6l 

195 

103 

83 
142 

131 

Total 
Ex.  9:7 
Deviation 

597 

594 

3 

459 
462 

—3 

LINKAGE  OF  DEFECTIVES  WITH  EACH  OTHER 

In  most  varieties  of  maize  there  are  ten  pairs  of  chromosomes. 
(Kuwada  191 5,  Kiesselbach  and  Petersen  1925.)  Therefore,  in 
crossing  thirteen  different  factors  in  all  combinations  some  cases 
of  linkage  are  almost  certain  to  occur.  The  difficulty  lies  in  their 
detection.  With  independent  inheritance  two  defectives  when 
crossed  give  a  9  7  ratio  in  F2.  With  complete  linkage  these  two 
defectives  should  give  a  1  :i  ratio.     Thus  the  entire  rangfe  of  cross- 


ENDOSPERM    CHARACTERS    IN    MAIZE 


555 


50 

49 

a  48 

P       An 

fl     47 

8 

o 

%     46 
I 

45 

44 

43.75 

y»1 

10 


20 


30 


40 


50 


PERCENTAGE  OP  CROSSING  OVER 

Fig.  64. — Diagram  showing  how  linkage  between  two  defective  seed 
factors  would  distort  the  normal  9:7  di-hybrid  ratio.  If  each  defective 
is  completely  linked  with  the  dominant  allelomorph  of  the  other,  a  1  :i  ratio 
is  expected. 

ing  over  from  50  per  cent  to  o  corresponds  to  a  range  of  43.75  to 
50  in  the  percentage  of  defectives.     This  is  shown  diagrammatic- 


556 


CONNECTICUT    EXPERIMENT    STATION 


BULLETIN    279 


ally  in  Figure  64.  Since  deviations  of  two  and  three  per  cent  are 
expected  to  occur  fairly  frequently  by  chance  alone,  the  difficulty 
of  detecting  any  but  the  closest  linkages  is  at  once  apparent. 
However,  the  fact  that  defectives  often  show  a  deficiency  and  very 


100 


2      80 


a 

m 
& 
o 

p^ 
o 

s 

u 

8 

« 


44.4 


PERCENTAGE  OF  CROSSING  OVER 

Fig.  65. — Diagram  showing  how  linkage  between  two  lethal  factors 
brought  in  from  opposite  parents  causes  an  increase  in  the  proportion  of 
heterozygotes. 


rarely  an  excess,  makes  it  necessary  to  regard  any  di-hybrid  ears 
which  produce  more  than  43.75  per  cent  defectives  as  possible 
cases  of  linkage.  Further  evidence  must  then  be  obtained  by 
growing  additional  di-hybrid  ears  or  by  examining  F3  progenies. 
If  the  high  percentage  of  defectives  in  F2  is  due  to  linkage,  then 


ENDOSPERM    CHARACTERS    IN    MAIZE  55  7 

the  F3  should  produce  an  excess  of  heterozygous  plants  as  well 
as  an  excess  of  recessive  seeds  on  a  majority  of  the  di-hybrid 
ears.  In  other  words,  when  two  defectives,  whose  factors  occupy 
loci  on  homologous  chromosomes,  are  brought  together,  a  condi- 
tion of  balanced  lethals  is  automatically  set  up.  With  complete 
linkage  these  two  lethals,  when  once  brought  together,  can  never 
be  separated  and  only  di-hybrid  ears  all  of  which  give  I  :i  ratios 
will  be  produced  thereafter.  The  diagram  in  Figure  65  shows 
how,  with  such  a  condition  of  balanced  lethals,  the  proportion  of 
double  heterozygotes  in  F3  is  expected  to  increase  with  the 
intensity  of  the  linkage. 

Balanced  lethals  were  first  suggested  by  Renner  (1916)  as  a 
possible  explanation  of  some  of  the  peculiar  results  from  the 
breeding  experiments  with  Oenothera.  De  Vries  (1916)  adopted 
the  explanation  to  account  for  the  production  of  twin  hybrids 
in  crosses  of  this  species,  but  he  failed  to  appreciate  the  full  signifi- 
cance of  the  effects  of  lethals  in  a  balanced  condition.  It 
remained  for  Muller  (1918)  in  his  classic  contribution  on  the 
inheritance  of  the  beaded  wing  character  in  Drosophila  to  show 
the  bearing  of  balanced  lethals  on  constant  hybridity,  the  sporadic 
appearance  of  certain  "mutants"  due  to  crossing-over,  and  the 
production  of  twin  hybrids. 

Of  the  59  crosses  shown  in  Figure  54,  in  which  di-hybrid  ears 
have  been  obtained,  those  which  have  given  marked  excesses  of 
defective  seeds  are  shown  in  Table  14. 

Table  14.     Crosses    of    Defective    Seeds   which    Produced    an    Excess    of 

Recessives  in  F2. 


Cross 

Normal 

Defective 

%   Defective 

dew  x  dez 

418 

376 

474 

dea  x  de5 

270 

235 

46.5 

deio  x  de« 

64 

59 

48.0 

den  x  den 

275 

251 

47-7 

F3  progenies  of  only  one  of  these  crosses,  de10  x  dez,  have  been 
grown.  Twelve  self-pollinated  F3  progenies  were  obtained  of 
which  five  were  segregating  for  both  types  of  defective  seeds. 
Although  the  di-hybrid  ears  are  not  in  excess,  as  would  be  expected 
under  balanced  lethal  conditions,  the  sample  is  too  small  to  permit 
any  final  conclusion  on  this  point.  When  the  normal  and  defec- 
tive seeds  on  these  five  di-hybrid  ears  are  counted,  it  is  found  that 
there  is  again  an  excess  of  the  recessive  seeds  as  is  shown  in 
Table  15. 

When  these  five  progenies  are  combined  with  the  two  F2  prog- 
enies already  shown,  making  a  total  of  1060  normal  to  916  defec- 
tives, the  deviation  is  52  ±  13.  The  average  percentage  of  defec- 
tive seeds  is  found  to  be  46.3,  which  indicates  linkage  with  cross- 
ing over  of  38.5  per  cent. 


55^  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

Table  15.     Normal  and  Defective  Seeds   from  F3   Progenies  of  a  Cross 
of  deio  x  dez  Indicating  Linkage  between  These  Factors. 


Ear  No. 

Normal 

Defective 

%   Defective 

858 

112 

99 

46.9 

859 

130 

100 

43-5 

862 

132 

112 

45-9 

867 

125 

105 

45-7 

868 

143 

124 

46.4 

Total 

642 

540 

45-7 

Expected 

665 

517 

43-7 

Deviation 

—23 

P.  E.  =  11.5 

It  should  be  mentioned,  that  with  a  condition  of  balanced 
lethals,  occasional  progenies  are  expected  in  F3  in  which  there  is 
a  deficiency  of  recessives  instead  of  an  excess.  This  condition 
would  be  brought  about  through  crossing  over  so  that  the  two 
lethal  factors,  originally  on  homologous  chromosomes,  are  now 
borne  on  the  same  chromosome.  Thus  unless  the  linkage  between 
the  two  lethals  were  fairly  close,  so  that  the  excesses  and  deficien- 
cies within  each  progeny  were  sufficient  to  permit  a  separation,  the 
two  types  of  F3  progenies  would  tend  to  balance  each  other  and 
linkage  would  be  almost  impossible  to  detect. 

LINKAGE  OF  DEFECTIVES  WITH  GROWTH  FACTORS 

The  method  of  improving  corn  by  selection  in  self-fertilized 
lines  aims  at  the  removal  of  all  recessive  abnormalities  such  as 
white  seedlings  and  defective  seeds.  There  seems  to  be  a  general 
belief  that  these  factors  have  a  deleterious  effect,  even  in  the 
heterozygous  condition. 

Lindstrom  (1920)  suggests  that  these  recessive  abnormalities, 
if  they  do  have  an  unfavorable  effect  in  the  heterozygous  condi- 
tion, are  permitted  to  persist  in  the  germplasm  only  when  they  are 
linked  with  particularly  good  growth  factors,  and  that  in  remov- 
ing them  by  inbreeding,  some  of  the  best  germplasm  is  lost. 
Jones  and  Mangelsdorf  (1925)  have  shown,  however,  that  inbred 
strains  from  which  all  recessive  abnormalities  have  been  eliminated, 
yield  fully  as  well  as  sister  strains  which  still  carry  one  or  more  of 
these  abnormal  characters.  Apparently  nothing  of  value  was  lost 
through  their  elimination ;  neither  was  there  any  marked  improve- 
ment when  their  supposedly  unfavorable  influence  was  removed. 
Still  assuming  that  these  factors  have  an  influence  in  the  heterozy- 
gous state,  a  probable  explanation  of  these  conflicting  results  is 
that  the  defective  seeds  and  other  lethal  abnormalities  are  per- 
mitted to  persist  and  accumulate,  not  because  they  are  linked  with 
especially  good  factors  for  development,  as  Lindstrom  has  sug- 
gested, but  because  their  presence  tends  to  keep  short  sections  of 
the  chromosomes  which  they  occupy  in  a  continued  state  of  hetero- 


ENDOSPERM    CHARACTERS    IN    MAIZE  559 

zygosity.  The  increased  vigor  which  results  from  such  enforced 
heterozygosity  of  the  accompanying  growth  factors  enables  the 
recessive  abnormalities  to  survive  in  the  germplasm  even  though 
they  have  an  unfavorable  influence  in  themselves. 

Furthermore,  when  two  such  lethal  factors  which  occupy 
homologous  chromosomes  are  brought  together,  a  condition  of 
balanced  lethals  is  set  up  which  may  so  increase  the  vigor  of  the 
stock  by  keeping  whole  chromosomes  or  large  sections  of  chromo- 
somes in  a  continued  state  of  heterozygosity,  that  the  lethals  are 
actually  given  an  advantage  and  are  able  to  survive  even  though 
they  are  linked  with  especially  poor  growth  factors  instead  of 
particularly  good  ones. 

Shull  (1923)  has  pointed  out  that  varieties  of  Oenothera  which 
carry  lethal  factors  are,  in  general,  more  vigorous  than  those 
which  lack  these  characters.  The  mechanism  of  crossing  over  in 
Oenothera  appears  to  be  different  from  that  in  most  species  as  is 
shown  by  both  cytological  and  genetic  studies;  Shull  (1923), 
Cleland  (1925).  All  of  the  characters  so  far  studied  in  this 
species  fall  into  a  single  linkage  group  and  the  amount  of  cross- 
ing over  between  the  members  of  the  group  is  relatively  low.  It 
is  possible,  therefore,  that  lethal  factors  in  Oenothera  keep  all  of 
the  chromosomes,  with  their  hereditary  factors  for  growth  and 
development,  in  a  continued  state  of  enforced  heterozygosity.  If 
such  is  the  case;  then  the  increased  vigor  brought  about  in 
Oenothera  by  the  presence  of  lethal  factors  is  probably  more 
marked  than  would  occur  in  other  species  where  there  are  as  many 
linkage  groups  as  chromosomes. 

A  PLANT  CHARACTER  FOR  DEFECTIVE  SEEDS 

In  addition  to  the  thirteen  endosperm  characters  which  cause 
one-fourth  of  the  seeds  on  segregating  ears  to  be  defective,  a 
plant  character  which  causes  defectiveness  in  all  the  seeds  on 
one-fourth  of  the  plants  has  been  found. 

This  character  appeared  in  the  de13  stock  which  was  received 
from  Mr.  H.  A.  Wallace  of  Des  Moines,  Iowa.  Mr.  Wallace 
had  found  among  the  plants  of  the  variety  "Illinois  Two  Stalk" 
several  which  produced  only  aborted  seeds,  and  which  appeared 
to  be  homozygous  for  defective  seeds.  Pollen  from  one  of  these 
plants  applied  to  a  hybrid  of  inbred  strains  known  to  be  free 
of  hereditary  defectives  produced  only  normal  seeds.  The  ¥1 
plants  grown  from  these  normal  seeds  were  selfed  and  produced 
some  ears  which  were  segregating  for  defective  seeds  of  the  type 
which  has  already  been  described  as  de13.  Not  all  of  the  ears 
were  segregating,  however,  as  should  have  been  the  case,  had  one 
of  the  parents  been  homozygous  for  the  defective  factor.  Nor 
did  the  recessive  seeds  on  the  segregating  F1  ears  resemble  the 


560  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    2/9 

aborted  seeds  of  the  pollen  parent.  The  extracted  recessives  on 
the  Fj  ears  were  completely  aborted,  appeared  to  have  no  endo- 
sperm tissue  and  showed  no  germination  whatever.  It  was  diffi- 
cult to  understand  how  this  type  could  have  been  obtained  in  a 
homozygous  condition,  or  why,  if  the  pollen  parent  was  homp- 
zygous  for  defective  seeds,  only  part  of  the  F1  ears  were 
segregating  for  the  character. 

This  confusing  situation  was  cleared  up,  however,  when  a  large 
number  of  F2  ears,  which  had  been  grown  for  another  purpose, 
were  harvested.  A  total  of  201  F2  ears  were  examined  and  of 
these  51,  or  almost  exactly  one-fourth,  bore  only  aborted  seeds  and 
were  identical  in  appearance  to  the  ears  of  the  grandparental 
pollen  parent.  The  other  150  ears  were  normal  in  appearance 
although  some  were  segregating  for  defective  seeds  and  others 
were  not. 

Apparently  the  plant  of  "Illinois  Two  Stalk"  which  served  as 
the  pollen  parent  for  this  cross,  in  addition  to  being  heterozygous 
for  a  recessive  endosperm  character  de13,  was  homozygous  for  a 
recessive  plant  character,  to  which  the  symbol  dep\  may  be  given. 
On  this  hypothesis  the  genetic  composition  of  the  parental  stocks 
and  the  F1  seeds  is  as  follows : 

Pollen  parent     De13  delz  dev\  dePi 
Seed  parent        Z><?13  Dex%  Dev\  Dev\ 
-r-,    c      ,  I  De1%  De^  Dev\  de^i 

**  beeds  {De»  dels  Devx  dev\ 

Half  of  the  Fx  seeds  when  selfed  should  produce  ears  segre- 
gating for  the  endosperm  character  de13.  The  other  half  should 
give  only  normal  seeds.  Apparently  these  conditions  have  been 
met;  of  the  five  Ft  ears  which  had  been  selfed  3  were  segregating 
for  defective  seeds  and  two  were  not. 

That  the  new  variation  de  v  is  actually  a  plant  character  is 
further  demonstrated  by  the  fact  that  the  recessive  plants  produce 
only  defective  seeds  regardless  of  the  pollen  which  they  receive. 
If  it  were  an  endosperm  character  cross  pollination  with  normal 
plants  should  give  only  normal  seeds. 

The  seeds  on  the  defective  ears  show  considerable  variation  in 
development,  some  being  almost  completely  defective,  others  only 
partially  so.  None,  however,  are  as  fully  developed  as  normal 
seeds,  though  many  are  capable  of  germination  and  when  grown 
produce  fairly  vigorous  plants  which  in  turn  bear  only  defective 
ears.  Defective  ears  from  such  homozygous  plants  are  shown  in 
Figure  66. 

There  seems  to  be  no  relation  between  the  defective  seed  type 
which  appeared  in  the  F2  endosperm  generation  and  the  defective 
ears  which  came  to  light  in  the  F2  plant  generation.  The  fact 
that  both  came  out  of  the  same  cross  is  regarded  merely  as  a 


ENDOSPERM    CHARACTERS    IN    MAIZE 


561 


coincidence.  The  two  have  now  been  separated  and  stocks  segre- 
gating- for  one  and  lacking  the  other  have  been  obtained. 

This  variation  is  of  interest  in  connection  with  the  defective 
seeds  because  it  produces  practically  the  same  effect  in  the  individ- 
ual seeds  as  do  some  of  the  endosperm  characters. 

The  character  of  the  endosperm  is  usually  determined  by  the 
genetic  constitution  of  the  zygote*  which  results  from  the  fusion 


Fig.  66. — Normal  and  defective  ears  of  the  depi  stock.  This  factor 
causes  all  of  the  seeds  on  one-fourth  of  the  plants'  to  be  defective 
regardless  of  the  genetic  constitution  of  the  endosperm. 


of  the  endosperm  nuclei  with  a  sperm  nucleus.  The  defective 
ear,  however,  represents  a  condition  in  which  the  expression  of  the 
hereditary  factors  of  the  new  sporophyte  is  prevented.  Appar- 
ently there  is  present  or  lacking,  in  the  recessive  plants,  something 
which  causes  all  of  their  seeds  to  be  aborted  regardless  of  the 
genetic  constitution  of  the  seeds  themselves.  Such  a  situation 
might  be  compared  to  a  population  of  plants  in  which  all  the 
plants  were  dwarfed  because  of  a  lack  of  moisture  or  fertility, 


*  To  be  strictly  accurate,  perhaps  the  term  "zygote"  ought  not  to  be  applied 
to  the  endosperm,  but  its  use  in  this  connection  is  probably  less  confusing 
than  would  be  the  adoption  of  a  new  term. 


562  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

while  in  a  better  environment  some  of  the  plants  would  be  tall 
and  others  dwarfed,  because  of  the  hereditary  factors  which  they 
carried. 

DISCUSSION 

The  fact  that  defective  seeds  have  been  noted  in  almost  every 
variety  of  maize  which  has  been  examined  and  that  only  in  one 
case  have  two,  from  different  varieties,  been  found  which  have 
proven  to  be  genetically  alike,  gives  some  indication  of  the  enor- 
mous number  of  these  characters  which  probably  exist.  The 
appearance  of  defective  seeds  in  a  homozygous  inbred  strain,  as 
the  result  of  mutation,  four  separate  times  in  four  years,  furnishes 
a  hint  as  to  their  probable  origin.  The  histological  studies  indi- 
cate that  these  characters  do  not  affect  specifically  the  endosperm 
or  embryo.  They  are,  apparently,  merely  variations  which  are 
extremely  deleterious  in  their  influence  and  their  major  effect  is 
seen  in  the  endosperm  generation  only  because  the  period  between 
fertilisation  and  the  maturity  of  the  seed  provides  the  first  oppor- 
tunity for  their  expression.  There  is  some  indication  that  these 
factors  also'  have  an  unfavorable  influence  in  the  heterozygous 
condition  and  the  fact  that  they  survive  and  accumulate  in  the 
germplasm  may  imply  that  they  tend  to  keep  other  factors,  which 
affect  development,  in  a  continued  state  of  heterozygosity. 

From  the  economic  standpoint  these  characters  are  probably  of 
no  direct  importance.  From  the  standpoint  of  the  geneticist  they 
offer  a  vast  amount  of  new  material  which  may  prove  useful  in 
charting  the  germplasm  of  this  species. 

Summary  Part  I 

1.  Defective  seeds  are  lethal  or  semi-lethal  characters  which 
affect  the  normal  development  of  the  seed. 

2.  These  characters  have  been  noted  in  many  varieties  of 
maize  and  it  is  estimated  that  one  plant  in  every  thirty,  on  the 
average,  is  heterozygous  for  defective  seeds. 

3.  Defective  seeds  have  been  the  most  frequent  variations  to 
arise  by  mutation  in  homozygous  inbred  strains  of  maize. 

4.  Fourteen  stocks  segregating  for  defective  seeds  have  been 
crossed  in  82  combinations.  Only  two  of  these  which  are  gene- 
tically alike  have  been  'found. 

5.  Histological  studies  show  that  normal  fertilization  appar- 
ently occurs  in  the  formation  of  these  seeds,  and  that  both  endo- 
sperm and  embryo  are  produced.  These  structures  develop  very 
slowly,  however,  and  remain  rudimentary.  Both  are  affected  to 
almost  the  same  degree  by  the  influence  of  the  lethal  factors. 

_  6.     The  relative  development  of  the  fourteen  stocks  of  defec- 
tive seeds  as  compared  to  normal  seeds  on  the  same  ears  ranges 


ENDOSPERM    CHARACTERS    IN    MAIZE  563 

from  2.4  per  cent  to  59  per  cent,  based  upon  the  dry  weight  of 
the  seeds. 

7.  The  following  indications  of  linkage  have  been  encountered : 
The  de2  factor  with  a  factor  for  white  seedlings,  probably  the 

tVo  factor,  which  is  a  representative  of  the  second  linkage  group. 
Crossing  over  is  about  1 1  per  cent. 

The  de1  and  deG  factors  with  the  factor  for  sugary  endosperm 
which  is  a  representative  of  the  third  linkage  group.  Crossing 
over  in  the  first  case  is  39  per  cent,  in  the  second  26  per  cent. 

Linkage  between  two  different  defectives  is  indicated  in  four 
crosses. 

8.  In  addition  to  the  1 3  endosperm  characters  which  cause  one- 
fourth  of  the  seeds  to  be  defective,  a  plant  character  which  causes 
all  of  the  seeds  on  one-fourth  of  the  plants  to  be  defective,  has 
been  found. 


564  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

PART    II 

No n- Hereditary  Defective  Seeds 

Almost  every  ear  of  maize  bears  some  aborted  seeds.  On  most 
ears  only  a  few  of  these  are  found  scattered  at  random  through- 
out the  inflorescence ;  on  others,  entire  regions  of  the  ear  produce 
only  defective  kernels.  These  undeveloped  seeds  have  commonly 
been  attributed  to  "imperfect  pollination"  though  it  has  never 
been  clear  just  what  conditions  were  included  by  this  term.  It  is 
now  evident  that  many  of  these  abortive  seeds  are  lethal  or  semi- 
lethal  characters,  inherited  as  simple  Mendelian  recessives,  but  it 
must  also  be  recognized  that  there  are,  in  addition  to  the  hered- 
itary characters,  various  other  types  of  defective  seeds  which  do 
not  appear  to  have  an  hereditary  basis  or  at  any  rate  are  not  in- 
herited as  recessive  endosperm  characters. 

These  non-hereditary  types  are  formed  regardless  of  whether 
the  ear  is  self-pollinated  or  cross-pollinated.  They  are  found  in 
all  varieties  and  occur  as  frequently  in  homozygous  inbred 
strains  as  in  ordinary  heterozygous  varieties.  In  appearance 
they  are  practically  identical  to  some  of  the  hereditary  types, 
and  in  separating  dominant  and  recessive  individuals  from 
a  segregating  ear,  they  are  included  with  the  latter.  Because  they 
sometimes  represent  a  significant  source  of  error  in  classification, 
it  has  been  considered  important  to  determine  the  frequency  of 
their  occurrence,  the  conditions  causing  their  production  and,  if 
possible,  some  means  of  distinguishing  them  from  the  Mendelian 
characters  which  they  resemble  so  closely. 

A  microscopic  examination  of  sectioned  material  from  various 
sources  showed  that  there  are  at  least  four  morphologically  dis- 
tinct types  of  non-hereditary  defective  seeds  and  that  these  are 
probably  produced  as  the  results  of  the  following  conditions : 

1.  Stimulus  resulting  from  pollination  without  fertilization. 
( Parthenocarpy. ) 

2.  Arrested  development  due  to  competition,  dominance  or 
other  physiological  conditions. 

3.  Irregularities  in  the  mechanism  of  fertilization. 

parthenocarpic  defectives 

The  first  of  these  four  types  was  found  on  an  ear  which  had 
been  bagged  as  a  pollination  check.  After  a  period  of  several 
weeks,  the  silks  grew  to  such  a  length  that  some  of  them  pro- 
truded from  below  the  bag  and  became  exposed  to  pollen.  As  a 
result,  a  few  of  the  ovules  on  this  ear  developed  slightly.  These 
were  not  normal  in  appearance  and  contained,  instead  of  the  usual- 
milky  fluid,  only  a  clear  watery  liquid  and  a  jellylike  tissue. 


ENDOSPERM    CHARACTERS    IN    MAIZE  565 

Microscopic  examination  of  sections  of  these  ovules  showed 
that  their  development  was  due  entirely  to  a  marked  growth  of 
nucellus.  The  embryo  sac  was  readily  recognized,  though  it  had 
begun  to  disintegrate  at  the  apical  end  and  the  antipodals  had 
moved  into  the  nucellar  tissue.  Within  the  sac  was  found  some 
disintegrated  granular  material  and,  in  several  specimens,  the  egg 
and  polar  nuclei  were  still  visible.  No  indications  of  fertilization 
were  found  in  any  of  the  sections  and  no  remains  of  the  pollen 
tube  could  be  distinguished,  although  these  might  have  been 
present  and  not  have  been  clearly  brought  out  by  the  stain  used. 
In  no  case  was  there  the  slightest  trace  of  endosperm  or  embryo. 
The  nucellus  in  maize  is  very  distinct  from,  the  endosperm  and 
there  is  little  danger  of  confusing  these  two  tissues.  In  none  of 
the  specimens  examined  did  the  nucellus  show  any  indication  of 
starch  grain  formation.  A  photomicrograph  of  one  of  these 
ovules  is  shown  in  Fig.  4,  Plate  XXVI. 

The  natural  conclusion  is  that  no  fertilization  occurred  in  these 
partially  developed  ovules.  Either  the  pollen  tubes  failed  to 
reach  the  micropyle,  or  if  they  reached  it,  failed  to  accomplish 
fertilization.  Apparently,  however,  the  tubes  in  growing  down 
the  styles  had  in  some  way  transmitted  a  stimulus  to  the  ovule 
which  resulted  in  a  marked  growth  of  the  nucellus  and  pericarp, 
even  though  fertilization  did  not  occur. 

Though  the  nucellus  of  maize  is  of  minor  importance  in  the 
mature  caryopsis,  becoming  compressed  into  a  thin  integument  by 
the  pressure  of  the  growing  endosperm,  yet  these  partially 
developed,  though  unfertilized,  ovules  are  comparable  in  certain 
respects  to  some  of  the  seedless  fruits  which  are  produced  in  the 
absence  of  pollination,  and  to  which  the  term  "parthenocarpic" 
has  been  applied. 

The  term  "parthenocarpy"  was  first  used  by  Noll  (1902)  to 
describe  the  situation  in  which  certain  plants,  under  exclusion  of 
pollen,  are  able  to  form  fruits  outwardly  normal,  or  nearly  so, 
but  in  which  the  seeds  are  absent  or  aborted.  Noll  reported 
this  condition  in  the  cucumber  and  Ewert  (1909,  1910)  has  noted 
it  in  other  fruits. 

Winkler  (1908)  makes  a  distinction  between  "stimulative" 
parthenocarpy  in  which  the  seedless  fruits  are  produced  only 
after  pollination  with  their  own  or  foreign  pollen  or  in  conse- 
quence of  an  insect  prick  or  other  irritation,  and  "vegetative" 
parthenocarpy  in  which  the  seedless  fruits  occur  without  the  action 
of  pollen  or  other  stimuli. 

Wellington  (1913)  found  that  in  Nicotiana  species,  capsules 
were  caused  to  swell  by  merely  tickling  them  with  a  sharp-pointed 
instrument.  Abortive  seeds,  probably  without  embryos,  were 
produced  by  singeing  the  buds  with  a  hot  wire,  by  exposure  of 
the  plants  to  chloroform  gas,  and  by  cutting  away  a  portion  of  the 


566  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    2/9 

pistil  and  pollinating  the  stub,  or  by  grafting  the  stigmatic  end  of 
another  pistil  to  the  excised  one  and  pollinating  the  new  pistil. 

These  abortive  ovules  of  Nicotiana,  as  well  as  those  of  maize, 
if  they  can  be  legitimately  termed  parthenocarpic,  fall  into  the 
category  of  "stimulative"  parthenocarpy  since  they  represent  a 
development  brought  about  by  stimuli  other  than  fertilization. 

An  inflorescence  of  maize  from  which  pollen  has  been  excluded 
throughout  the  season  is  shown  in  Fig.  67.  Maize  never  produces 
seeds  of  any  description  in  the  complete  absence  of  pollen  though 
it  may  be  possible  to  produce  with  artificial  stimuli,  such  as  used 
by  Wellington  in  Nicotiana,  the  same  development  of  the  ovules 
which  results  when  pollen  tubes  enter  the  style  without  accom- 
plishing fertilization. 

Parthenocarpy  is  not  to  be  confused  with  parthenogenesis.  In 
the  former  no  embryo  is  produced,  in  the  latter  normal  seed  for- 
mation may  occur.  Parthenogenesis  occurs  regularly  in  some 
plants  but,  so  far  as  is  known,  has  never  been  found  in  maize  or 
closely  related  species.  Parthenocarpy,  on  the  other  hand,  may  be 
widely  distributed.  In  species  such  as  maize,  however,  where 
the  stimulated  parts  are  of  little  importance,  parthenocarpy  is  of 
significance  only  as  it  has  a  bearing  on  the  physiology  of  pollina- 
tion, or  represents  a  source  of  error  in  genetic  experiments. 


SOME   CONDITIONS    WHICH    INFLUENCE    THE   FREQUENCY   OF 
PARTHENOCARPIC  DEFECTIVES   IN   MAIZE 

Several  conditions  noted  in  the  experience  of  making  artificial 
pollinations  in  maize  suggested  the  possibility  that  the  frequency 
with  which  parthenocarpic  defectives  are  produced  probably 
depends  to  a  large  extent  on  the  age  of  the  silks.  As  already  men- 
tioned, these  seeds  were  first  noted  on  ears  which  had  received  no 
pollen  until  several  weeks  after  the  silks  had  emerged.  An  inbred 
strain  of  flint  corn  in  which  the  husks  are  extremely  long,  so  that 
the  silks  must  attain  a  length  of  50  cm.  or  more  before  emerging, 
regularly  produces  a  large  proportion  of  parthenocarpic  defectives. 
This  same  strain  bears  normal  seeds  when  the  husks  are  cut  back. 

These  conditions  suggest  that  the  silks  may  become  of  such  a 
length  that  the  pollen  tubes  are  no  longer  capable  of  reaching  the 
micropyle  or  that  the  embryo  sac,  after  lying  idle  for  so  long  a 
period,  becomes  disorganized  and  is  no  longer  capable  of  entering 
into  fertilization  even  though  the  styles  still  retain  their  recep- 
tivity and  furnish  a  medium  for  the  growth  of  the  pollen  tubes. 

Parthenocarpic  defectives  are  also  frequently  produced  on  ears 
which  have  been  artificially  pollinated  several  days  sooner  than  the 
normal  time.  In  making  hand  pollinations  it  is  sometimes  neces- 
sary to  use  ears  from  which  the  silks  have  not  yet  emerged. 
The  husks  are  cut  back  to  the  tip  of  the  spike,  exposing  silks  which 
under  natural  conditions  would  not  have   been   pollinated   until 


ENDOSPERM    CHARACTERS    IN    MAIZE 


567 


I 


mm 


~~\z 


•:•    ■ 


Fig.  67. — An  inflorescence  from  which  pollen  has  been 
excluded  throughout  the  season,  compared  to  an  open-polli- 
nated ear  from  the  same  strain.  No  development  of  any  kind 
occurs  in  the  complete  absence  of  pollination. 


568  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

several  days  later.  On  ears  treated  in  this  manner  it  is  not  un- 
common for  the  ovules  at  the  tip  to  be  parthenocarpic,  suggesting 
that  the  silks  are  capable  of  receiving  pollen  before  the  embryo  sac 
is  ready  for  fertilization. 


INFLUENCE  OF  AGE  OF  SILKS 

To  determine  the  influence  of  the  condition  of  the  silks  on  the 
frequency  of  parthenocarpy,  the  following  experiments  were 
made:  The  husks  were  removed  from  young  ear  shoots  at 
periods  of  about  six,  four,  and  two  days  before  the  time  at  which 
the  ears  would  normally  have  silked  out.  The  removal  of  the 
husks  and  the  surrounding  leaf  sheath  so  weakened  the  stalk  at  the 
ear-bearing  node  that  it  was  necessary  to  brace  the  plants  by 
tying  them  to  a  thin  strip  of  wood  above  and  below  this  node. 

These  immature  inflorescences  were  pollinated  with  normal 
pollen  from  earlier  plants  and  were  covered  with  paper  bags  after 
pollination  to  avoid  further  exposure.  As  a  check,  ears  opened 
at  an  early  stage  were  covered  with  paper  bags  but  were  not  polli- 
nated until  several  days  later,  or  at  about  the  time  that  they  would 
have  received  pollen  under  natural  conditions. 

The  spikes  at  six  days  before  silking  were  very  small,  and  had 
only  a  few  rudimentary  silks,  without  lateral  hairs,  at  the  base. 
Spikes  at  four  days  before  flowering  time  had  short  but  well 
developed  silks  at  the  base  and  rudimentary  hairless  silks  at  the 
tip.  At  two  days  before  blooming  time  the  spikes  were  well 
covered  with  silks. 

The  normal  blooming  time  for  all  of  these  plants  was  deter- 
mined from  sister  plants  of  the  same  first  generation  hybrid. 
Throughout  these  experiments  only  plants  of  a  first  generation 
hybrid  between  two  inbred  strains  were  used.  These  strains  were 
free  from  any  types  of  hereditary  defective  seeds  and  were  homo- 
zygous for  all  visible  characters.  Consequently  the  plants  of  the 
Fx  hybrid  were,  for  all  practical  purposes,  genetically  identical 
and  any  difference  in  the  number  of  defective  seeds  was  caused 
by  influences  other  than  heredity.  Under  uniform  soil  condi- 
tions all  plants  of  this  hybrid  come  into  silk  at  about  the  same 
time,  making  it  possible  to  determine  fairly  accurately  the  normal 
blooming  time  of  the  plants  which  were  prematurely  pollinated. 

In  addition  to  the  ears  which  were  pollinated  ahead  of  time, 
another  series  was  bagged  and  pollinated  at  the  normal  blooming 
time  and  at  periods  of  7,  17  and  25  days  later.  The  results  of 
these  premature  and  delayed  pollinations  are  given  in  Table  16. 
The  seeds  from  only  a  single  ear  are  counted  in  each  case.  In 
the  pollinations  made  at  six  days  before  blooming  time  no  seeds 
were  set  and  in  that  made  at  25  days  after  silking  all  the  ears 


ENDOSPERM    CHARACTERS    IN    MAIZE  569 


Table  16. 


Influence  of  the  Age  of  Silks 

upon  the  Percentage  of  Parthe 

nocarpic  Defectives. 

Days  before  or          Total   No. 

No.  of 

Percent 

after  Silking            of  Seeds 

Defectives 

Defective 

6  days  before              0 

O 

0 

4      "                           237 

85 

35-9 

2      "                            121 

8 

6.6 

Normal  time            783 

8 

1.0 

7  days  after             678 

11 

1.6 

17     "                         382 

83 

21.7 

25      "                            60 

10 

16.7 

except  one  were  barren.  The  one  exception  produced  60  poorly 
developed  seeds  at  the  tip  of  the  spike  of  which  50  contained 
endosperm  and  embryo  and  ten  were  parthenocarpic. 

These  results  indicate  that  the  silks  are  apparently  receptive  to 
pollen  for  a  period  greater  than  that  during  which  fertilization  can 
occur,  and  that  pollinations  which  are  made  very  early  or  very 
late  do  not  accomplish  fertilization  in  many  cases  but  succeed 
merely  in  inducing  a  development  of  the  nucellus  and  pericarp. 


INFLUENCE  OF  THE  AGE  OF  POLLEN 

An  experiment  was  also  made  to  determine  whether  the  condi- 
tion of  the  pollen  has  any  influence  on  the  proportion  of  partheno- 
carpic defectives  which  are  produced. 

This  experiment  was  planned  to  test  the  pollen  at  regular  inter- 
vals of  six  hours  after  collection.  In  order  to  avoid  the  necessity 
of  making  some  of  the  pollinations  during  the  night,  two  collec- 
tions were  made.  The  first,  designated  as  A  in  the  table,  was 
made  at  noon,  the  second,  B,  from  the  same  plants  in  the  evening, 
about  seven  hours  later. 

The  pollen  was  kept  in  a  cool  basement  room  where  the  temper- 
ature and  humidity  were  relatively  constant.  Pollinations  with 
the  two  lots  were  made  every  twelve  hours  on  silks  which  were 
trimmed  off,  as  nearly  as  possible,  to  a  uniform  length,  and  a 
supply  of  pollen  ample  to  insure  full  ears  under  natural  conditions 
was  applied.  Three  ears  were  pollinated  for  each  six  hour  period 
up  to  44  hours,  which,  from  previous  experience,  was  considered 
the  maximum  period  of  viability  for  maize  pollen. 

When  the  ears  were  harvested  a  marked  difference  was  noted 
in  those  resulting  from  the  two  lots  of  pollen.  Ears  produced 
from  pollination  with  Lot  A  were  completely  filled  with  the  excep- 
tion of  those  resulting  from  the  44  hour  pollen.  Ears  from  the 
B  lot  were  all  poorly  filled,  the  earlier  pollinations  showing  a  few 
missing  kernels,  the  later  ones,  many.  Apparently  the  pollen 
collected  in  the  evening  was  injured  in  some  way,  perhaps  by  the 
higher  humidity  of  the  atmosphere  at  that  time. 


57°  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

One  ear  from  each  pollination  was  shelled  and  the  normal  and 
defective  kernels  counted.     The  figures  are  given  in  Table  17. 

Table  17.     Influence  of  the  Age  of  Pollen  upon  the  Percentage  of  Parthe- 
nocarpic Defectives. 


Hours  after 
Collection 

Total  No. 
of  Seeds 

No.  of 
Defectives 

Percent 
Defective 

Lot  A 

7 
19 
3i 
44 

586 
641 
718 
S8S 

23 
19 
14 
46 

3-9 
3-0 
2.0 
7-9 

Lot  B 

0 

788 

47 

6.0 

13 

554 

51 

9.2 

24 
37 

543 
161 

57 
45 

10.5 
28.0 

It  is  noted  that  in  Lot  A,  where  there  was  apparently  an  abund- 
ance of  good  pollen  at  all  periods,  there  is  little  change  in  the 
percentage,  of  defective  seeds,  accompanying  the  increase  in  age 
of  the  pollen.  In  the  ears  of  Lot  B,  however,  where  the  number 
of  viable  pollen  grains  was  never  enough  to  give  completely  filled 
ears,  the  percentage  of  parthenocarpic  defectives  increased  pro- 
gressively with  the  age  of  the  pollen,  beginning  with  6.0  per  cent  at 
time  of  collection  and  ending  with  28.0  at  37  hours. 

There  seems  to  be  no  question  that  the  age  of  pollen  has  some 
influence  on  the  frequency  with  which  the  parthenocarpic  defec- 
tives occur.  It  is  probable  that  in  the  older  pollen  many  of  the 
grains  are  capable  of  germinating  but  are  not  vigorous  enough  to 
reach  the  micropyle,  and  succeed  merely  in  transmitting  a  stimulus 
which  induces  the  development  of  the  nucellus  and  pericarp. 
Where  there  is  an  abundance  of  pollen  these  weakened  pollen  tubes 
produce  no  effect  since  fertilization  can  be  accomplished  by  more 
vigorous  tubes  growing  in  the  same  styles.  Where  the  supply  of 
viable  pollen  is  limited,  however,  so  that  in  many  of  the  styles  there 
is  no  such  competition,  the  weakened  pollen  grains  germinate  but 
succeed  only  in  producing  parthenocarpic  defectives. 

There  are,  no  doubt,  other  conditions  which  influence  the  appear- 
ance of  parthenocarpic  defectives.  Any  unfavorable  weather 
conditions,  for  example,  which  might  prevent  the  pollen  tubes 
from  reaching  the  micropyle,  would  probably  result  in  the  forma- 
tion of  parthenocarpic  defectives.  In  artificially  self-pollinated 
ears  where  an  abundance  of  pollen  is  applied  to  ears  that  are  fully 
silked  out,  the  proportion  of  parthenocarpic  seeds  which  are  scat- 
tered throughout  the  ear  remains  relatively  constant.  In  ears  in 
which  it  is  evident  that  a  large  number  of  these  seeds  are  present 


ENDOSPERM    CHARACTERS    IN    MAIZE 


571 


Fig.  68. — Ears  which  have  been  pollinated  two  separate  times. 
The  first  pollination  resulted  in  normal  seeds ;  the  second,  made 
some  days  later,  produced  only  "arrested"  seeds. 


572  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

at  the  tip  it  has  been  the  custom  to  discard  this  area  of  the  in- 
florescence in  making  a  study  of  the  hereditary  defective  seeds. 


ARRESTED   DEVELOPMENT 

The  second  type  of  non-hereditary  defective  was  found  on  ears 
in  a  crossing  plot  where  the  pollen  parent  had  been  planted  at 
two  different  times.  The  first  lot  of  plants  to  come  into  tassel  had 
provided  pollen  enough  to  fertilize  only  a  few  of  the  ovules  on  the 
ears  of  the  de-tasselled  pistillate  plants.  When  the  second  plant- 
ing came  into  bloom  and  pollen  was  again  shed,  the  seeds  result- 
ing from  these  later  pollinations  were  very  small  and  abortive 
and  never  attained  a  normal  development.  They  differed,  in  gen- 
eral, from  the  parthenocarpic  defectives,  in  having  a  milky  opaque 
appearance  in  contrast  to  the  clear  watery  condition  of  the  latter, 
although  this  distinction  is  not  marked  enough  to  permit  an  accu- 
rate separation  of  these  two  types  on  the  same  ear. 

Sectioned  material  showed  that  these  seeds,  though  abortive  in 
appearance,  were  quite  normal  in  structure,  having  a  well  devel- 
oped embryo  and  an  endosperm  with  numerous  starch  grains  in 
the  cells.  Instead  of  growing  at  a  normal  rate,  however,  these 
seeds  grew  very  slowly  and  in  some  cases  ceased  their  develop- 
ment at  a  stage  which  corresponds  to  that  found  in  the  normal 
seed  at  about  ten  days  or  two  weeks  after  fertilization. 

Apparently  these  abortive  seeds  represent  an  arrested  develop- 
ment, caused  by  the  presence  of  more  advanced  seeds  on  the  same 
inflorescence.  This  may  be  merely  the  result  of  a  competitive 
effect  or  it  may  be  due  to  a  sort  of  "dominance"  which  the  earlier 
formed  seeds  have  over  the  physiological  processes  of  the  inflores- 
cence, similar  to  the  advantage  which  a  growing  tip  has  over  the 
lateral  branches  both  in  plants  and  in  lower  animals.  The  latter 
possibility  is  suggested  by  a  condition  which  is  sometimes  found 
on  ears  in  which  two  separate  hand  pollinations  have  been  made, 
the  first  when  only  a  few  silks  are  out,  and  the  second  some  days 
later  when  the  remaining  silks  have  appeared.  The  first  pollina- 
tion causes  the  formation  of  normal  seeds  at  the  base  of  the  ear, 
while  the  second  pollination  often  results  in  the  production  of 
"arrested"  seeds  over  the  remainder  of  the  ear.  Such  ears  are 
shown  in  Figure  68.  Here  the  results  do  not  appear  to  be  due  to 
competition  alone  since  the  competitive  effect  would  be  expected 
only  where  the  two  areas  meet.  Moreover,  the  same  plant  would 
be  capable  of  producing  normal  seeds  over  the  entire  ear  had  all 
the  silks  been  pollinated  simultaneously. 

In  this  case,  apparently,  the  first  seeds  to  develop  so  dominate 
the  physiological  processes  of  the  plant,  that  the  later  seeds  are 
deprived  of  normal  nourishment.  In  fact  it  is  not  uncommon  in 
such  ears  for  the  area  containing  the  arrested  seeds  to  be  com- 


ENDOSPERM    CHARACTERS    IN    MAIZE  573 

pletely  cut  off  so  that  the  upper  part  of  the  spike  disintegrates 
and  is  finally  pushed  off  completely. 

Seeds  of  the  arrested  type  are  not  an  important  source  of  error 
in  the  defective  seed  studies,  since  they  occur  most  commonly 
when  all  the  silks  are  not  pollinated  simultaneously.  In  some 
strains,  however,  they  occur  regularly  even  when  all  the  silks  are 
pollinated  at  once.  In  such  strains  due  allowance  must  be  made 
for  the  disturbance  which  they  cause  in  the  ratios. 

IRREGULARITIES   IN   THE  FERTILIZATION    MECHANISM 

•  The  non-hereditary  defectives  which  are  believed  to  be  due  to 
irregularities  in  the  fertilization  process  are  morphologically  of 
two  types.  The  first  of  these,  designated  as  "germless,"  contains 
an  endosperm  but  lacks  the  embryo,  while  the  second  type,  termed 
"miniature,"  has  both  structures  though  these  are  greatly  reduced 
in  size. 

In  the  germless  seeds  the  development  of  the  endosperm  varies 
from  a  condition  in  which  only  a  small  mass  of  mealy  tissue  occurs 
to  one  in  which  well  defined  floury  and  corneous  regions  are  dis- 
tinguishable. In  the  miniature  type  the  endosperm  and  embryo 
are  both  apparently  normal,  though  the  former  is  greatly  com- 
pressed by  the  pressure  of  the  normal  seeds  on  either  side  and  the 
latter  is  scarcely  half  the  size  of  a  normal  embryo. 

These  types  are  found  in  small  numbers  on  almost  any  ear  of 
maize.  A  count  of  the  seeds  on  fourteen  open-pollinated  ears  of 
a  hybrid  between  inbred  strains  known  to  be  free  from  hereditary 
defectives,  gave  total  of  10,235  seeds  of  which  50  or  approx- 
imately one  out  of  every  205  seeds  was  germless.  The  miniature 
seeds  on  nine  of  these  ears  were  also  counted  and  fifteen  were 
found  in  a  total  of  6,245  or  one  in  every  416  seeds.  The  germ- 
less seeds  apparently  occur  about  twice  as  frequently  as  the 
miniature  type. 

The  suggestion  that  these  two  types  of  non-hereditary  defec- 
tives, particularly  the  germless  type,  are  due  to  irregularities  in 
fertilization,  comes  from  a  microscopic  examination  of  sectioned 
material.  In  specimens  fixed  at  the  early  dough  stage,  it  was 
found  that  the  germless  seeds  contained  normal  endosperm  tissue 
and  an  aleurone  layer  but  that  no  trace  of  embryo  tissue  was 
present.  Opposite  the  micropyle  was  a  cavity  in  the  endosperm 
and  this  appeared  to  be  a  remnant  of  the  embryo  sac.  Within  the 
cavity  was  found,  in  two  specimens,  a  single  large  nucleus,  to  all 
appearances  the  unfertilized  or  undivided  egg,  or  perhaps  the 
polar  nucleus.  A  photomicrograph  of  one  of  these  specimens  is 
shown  in  Figure  3,  Plate  XXVI. 

Apparently  this  represents  a  case  of  single  fertilization,  in  which 
one  of  the  female  nuclei  had  never  been  fertilized  or,  if  fertilized, 


574  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

had  not  divided.  The  other  nucleus,  after  fusion,  or  as  the  result 
of  some  other  stimulus,  had  divided  and  produced  an  endosperm, 
which,  though  greatly  reduced  in  size,  was  normal  in  structural 
details,  the  cells  being  packed  with  starch  grains. 

There  is,  of  course,  the  possibility  that  fertilization  did  not  occur 
at  all ;  that  the  aborted  endosperm  was  produced  vegetatively  by 
the  division  of  the  polar  nuclei.  The  endosperm  of  maize  is 
usually  the  product  of  a  sexual  fusion  in  which  one  of  the  sperm 
from  the  pollen  tube  fuses  with  the  two  polar  nuclei  of  the 
embryo  sac.  In  gymnosperms,  however,  and  in  some  angio- 
sperms,  the  endosperm  is  formed  independently  of  any  fusion  and 
in  some  species,  where  fusion  naturally  occurs,  endosperm  forma- 
tion may  be  induced  by  artificial  stimuli. 

From  the  experience  of  artificial  pollinations  in  maize  it  can  be 
positively  stated  that  no  development  of  the  endosperm  occurs  in 
the  complete  absence  of  pollination.  However,  the  partheno- 
carpic  defectives,  already  described,  show  that  the  mere  growth  of 
the  pollen  tube  in  the  style,  transmits  a  stimulus  to  the  ovule  which 
causes  the  nucellus  to  expand  and  the  pericarp  to  grow.  It  would, 
therefore,  not  seem  to  be  an  impossibility  for  the  same  stimulus 
of  pollination  to  occasionally  induce  endosperm  formation  even 
though  actual  fertilization  did  not  occur.  In  other  words,  a  con- 
dition regularly  found  in  the  gymnosperms  and  some  of  the  angio- 
sperms,  might  be  encountered  occasionally  as  an  irregularity  in 
this  particular  angiosperm. 

That  the  endosperm  of  the  germless  seeds  is  not  produced 
vegetatively  by  the  division  of  one  or  both  of  the  unfertilized 
polar  nuclei,  is  shown  by  a  series  of  pollinations  in  which  domi- 
nant aleurone  and  endosperm  color  characters  are  introduced  by 
the  pollen  parent.  On  ears  of  a  variety  with  white  endosperm 
pollinated  by  one  in  which  the  endosperm  is  yellow,  all  of  the 
germless  seeds  with  sufficient  tissue  to  show  any  color,  were 
yellow.  On  ears  of  a  strain  with  colorless  aleurone,  pollinated  by 
a  type  with  purple  aleurone,  the  most  developed  of  the  germless 
seeds  were  purple.  The  appearance,  due  to  xenia,  of  these  dom- 
inant characters  from  the  pollen  parent,  leaves  no  doubt  that  the 
male  nucleus  has  taken  part  in  the  formation  of  the  endosperm 
tissue  of  these  aberrant  seeds  and  that  they  are  not  the  result  of  a 
vegetative  division  of  one  or  both  of  the  polar  nuclei. 

On  the  other  hand,  if  the  germless  seeds  are  due  to  single 
fertilization,  as  first  suggested,  it  seems  rather  strange  that  seeds 
containing  an  embryo,  but  lacking  the  endosperm,  are  not  found 
as  frequently  as  the  reciprocal  combination.  If  only  one  fertiliza- 
tion occurs,  the  fusion  of  egg  nucleus  and  sperm  might  be 
expected  to  take  place  as  frequently  as  that  of  polar  nuclei  and 
sperm,  since  under  normal  conditions  the  two  usually  occur  almost 
simultaneously.     (Weatherwax  1919,  Miller  1919.) 


ENDOSPERM    CHARACTERS    IN    MAIZE 


575 


There  is  the  possibility  that  seeds  of  this  type  do  occur  but  that 
the  embryo  is  so  dependent  upon  the  endosperm  for  nourishment 
that  it  fails  to  develop  in  the  absence  of  the  latter,  and  seeds 
resulting  from  a  fusion  of  sperm  and  egg"  nucleus  alone  are  classi- 
fied as  parthenocarpic  defectives. 

Another  possibility  is,  that  if  single  fertilization  occurs  at  all, 
it  always  results  in  formation  of  an  endosperm  regardless  of 
which  of  the  nuclei  of  the  embryo  sac  was  fertilized.  Such  a 
situation  would  be  in  interesting  contrast  to  other  species  where 
irregularities  of  this  sort  usually  result  in  the  production  of 
embryos.  In  Naias  major,  for  example,  Guignard  found  that  in 
some   cases   the   second   male   nucleus    fused   with   the   synergid 


Fig.  69. — Four  open-pollinated  ears  of  a  homozygous  inbred  strain 
showing  the  varying  proportions  of  non-hereditary  defective  seeds 
which  may  occur  under  natural  conditions. 

instead  of  the  primary  endosperm  nucleus.     When  this  occurred 
two  embryos  and  no  endosperm  were  produced. 

The  possibility  that  single  fertilization  always  results  in  endo- 
sperm formation  in  this  species,  is  offered  only  as  a  suggestion. 
It  seems  more  probable  that  seeds  with  embryos  but  lacking  an 
endosperm  are  so  poorly  developed  that  they  are  classed  as 
parthenocarpic  and  it  is  expected  that  such  specimens  will  be 
encountered  when  more  of  the  parthenocarpic  defectives  in  early 
stages  of  development  are  examined  microscopically. 


576  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

In  this  connection  it  may  be  mentioned  that  Harlan  and  Pope 
(1925)  have  recently  described  a  number  of  defective  seeds  in 
barley,  in  part  of  which  no  embryo  could  be  found,  in  the 
remainder  no  endosperm,  both  types  being  attributed  by  them  to 
single  fertilization.  In  the  so-called  "endosperm  deficient" 
kernels,  however,  disintegrating  masses  of  tissue  whose  cells  show 
early  stages  of  starch  grain  formation  are  found.  These  writers 
believe  this  tissue  to  be  of  nucellar  origin. 

In  the  parthenoearpic  defectives  of  maize,  in  which  the  nucellus 
is  stimulated  to  such  a  marked  degree,  no  evidence  of  starch  grain 
formation  has  been  found  in  any  stage.  Though  the  possibility 
of  the  nucellus  assuming  the  role  of  an  endosperm  under  some 
conditions  is  not  denied,  it  seems  more  probable,  from  the  descrip- 
tion given  by  these  writers,  and  from  their  photomicrographs,  that 
the  so-called  endosperm  deficient  kernels  of  barley  are  not  due  to 
single  fertilization  but  are  the  result  of  some  influence  which 
has  arrested  the  development  of  embryo  and  endosperm  alike. 
The  photographs  show  clearly  that  the  embryo  in  these  abortive 
seeds  of  barley  is  by  no  means  normal. 

Since  there  is  fairly  good  cytological  evidence  that  the  germless 
seeds- are  the  product  of  single  fertilization,  it  is  possible  that  the 
miniature  seeds  are  also  due  to  some  irregularity  in  the  mechanism 
of  fertilization,  perhaps  to  a  failure  of  the  antipodal  polar  nucleus 
to  fuse  with  the  micropylar  polar  nucleus.  Normally  the  sperm 
fuses  first  with  the  one  polar  nucleus  and  almost  immediately  the 
two  are  joined  by  the  second  polar  nucleus,  this  process  constitu- 
ting the  "triple  fusion"  characteristic  of  the  formation  of  the 
endosperm  in  many  angiosperms.  In  maize  the  two  polar  nuclei 
never  fuse  until  after  fertilization  (Miller  1919,  Weatherwax 
1919). 

It  is  conceivable  that  occasionally,  as  often  as  once  in  416  times, 
this  exceedingly  precise  mechanism  might ,  show  some  variation 
such  that  the  second  polar  nucleus  would  fail  to  enter  into  the 
fusion.  Theoretically  such  a  variation  would  result  in  formation 
of  embryo  and  endosperm,  though  the  latter,  being  the  product  of 
the  fusion  of  two  haploid  entities  instead  of  three  would  have  the 
diploid  instead  of  the  usual  triploid  number  of  chromosomes 
and  might  be  expected  to  be  somewhat  reduced  in  size,  as  a 
consequence. 

Such  a  condition  is  not  to  be  confused  with  that  suggested  by 
Webber  (1900)  as  a  possible  explanation  of  mosaic  seeds  in  which 
part  of  the  endosperm  shows  maternal  characters,  the  remainder 
paternal.  Webber  thought  that  these  seeds  might  be  due  to  a 
fusion  of  the  sperm  with  one  polar  nucleus  while  the  second 
polar  nucleus  divided  vegetatively,  the  two  tissues  growing  side 
by  side.  East  and  Hayes  (1911)  suggested  that  these  seeds  were 
due  to  a  vegetative  segregation,  and  Emerson  (T921)  has  recently 


ENDOSPERM    CHARACTERS    IN    MAIZE 


577 


presented  considerable  evidence  indicating-  that  the  mosaic  seeds 
are  the  result  of  non-disjunction  of  single  pairs  of  chromosomes. 


Fig.  70. — From  top  to  bottom:  normal,  miniature,  germless,  and 
parthenocarpic  defectives  from  same  ear.  The  miniature  seeds  con- 
tain both  endosperm  and  embryo,  the  germless  seeds  only  an  endo- 
sperm, while  the  parthenocarpic   defectives   lack  both  structures. 

The  suggested  expanation  of  the  miniature  seeds,  on  the  other 
hand,  is  that  one  of  the  polar  nuclei  is  fertilized  but  that  the  other, 


578  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

failing  to  fuse,  does  not  divide  and  plays  no  part  in  formation  of 
the  endosperm. 

Whether  or  not  the  miniature  and  germless  seeds  are  due  to  the 
irregularities  in  the  mechanism  leading  up  to  fertilization,  which 
have  been  suggested,  is  not  of  first  importance.  Such  a  precise 
mechanism  as  is  found  in  maize  might  be  expected  to  show  occa- 
sional accidental  variations,  and  such  variations  would  probably 
result  in  defective  seeds.  The  fact  of  main  importance,  how- 
ever, is  that  these  two  distinct  types  of  non-hereditary  defectives 
appear  with  a  regularity  that  makes  them  a  small,  though  con- 
stant, source  of  error  in  genetic  studies  of  the  hereditary  types 
which  they  resemble  so  closely. 

Summary — Part  II 

1.  Four  morphologically  distinct  types  of  non-hereditary 
defectives,  which  resemble  in  appearance  certain  of  the  hereditary 
types  discussed  in  Part  I,  have  been  found. 

2.  "Parthenocarpic  defectives  resemble  complete  defectives. 
They  contain  neither  endosperm  or  embryo  and  are  the  result 
of  a  marked  growth  of  the  nucellus  and  pericarp  following  polli- 
nation without  fertilization. 

3.  The  frequency  of  parthenocarpic  defectives  is  influenced  by 
the  age  of  silks,  age  of  pollen,  and  probably  by  other  environ- 
mental conditions. 

4.  "Arrested"  seed  contain  endosperm  and  embryo  but  both 
structures  are  retarded  in  their  development  due  to  competi- 
tion or  physiological  dominance  of  normal  seeds  on  the  same 
inflorescences. 

5.  "Germless"  seeds  contain  endosperm  tissue  but  no  embryo. 
Cytological  examinations  indicate  that  these  seeds  are  due  to 
single  fertilization  instead  of  the  usual  double  fertilization. 

6.  "Miniature"  seeds  are  normal  in  structure  though  some- 
what reduced  in  size.  It  is  suggested  that  they  are  the  result  of 
irregularities  in  fertilization  such  that  their  endosperm  is  diploid 
instead  of  triploid  in  its  chromosome  number. 


ENDOSPERM    CHARACTERS    IN    MAIZE  5  79 


PART  III 

Genetic  Factors  Which  Influence  the  Texture  of 
the  Endosperm 

In  addition  to  the  thirteen,  and  probably  many  more,  genetic 
factors  which  primarily  affect  the  amount  of  endosperm  pro- 
duced, there  are  a  number  of  others  which  govern  the  chemical 
or  physical  nature  of  the  storage  material  and  result  in  differences 
in  the  texture  of  the  endosperm. 

Several  of  these  factors  are  already  familiar  to  geneticists. 
Three  of  them,  sugary,  waxy,  and  shrunken,  are  inherited  as 
simple  Mendelian  recessives  and  are  complementary  in  their 
action,  crosses  between  any  two  of  these  types  resulting  in  starchy 
Fx  seeds. 

The  texture  of  the  endosperm  of  flint  and  flour  varieties  of  corn 
has  also  been  found  to  show  alternative  inheritance  in  some 
crosses.  These  characters  differ  from  the  other  three  in  that  a 
double  dose  of  one  is  always  dominant  to  a  single  dose  of  the 
other,  with  the  result  that  the  Fx  seeds  of  a  cross  are  always 
maternal  in  appearance  no  matter  which  way  the  cross  is  made, 
while  the  F2  progenies  show  a  i  :i  segregation  no  matter  which 
type  of  Fx  seeds  is  planted.     (Hayes  and  East,  1925.) 

To  these  already  familiar  characters  affecting  the  texture  of 
the  endosperm,  must  be  added  another  one,  brittle,  which  has 
appeared  in  the  course  of  these  investigations. 

BRITTLE   ENDOSPERM* 

In  1922,  in  a  lot  of  about  100  self-pollinated  ears  of  Sanford 
White,  a  typical  eight-rowed,  New  England  flint  variety,  were 
found  two  ears  which  segregated  for  an  endosperm  character 
which  had  not  been  noted  previously.  In  appearance  the  reces- 
sive seeds  on  these  two  ears  were  somewhat  similar  to  the  familiar 
sugary  seeds,  being  translucent  instead  of  opaque  and  wrinkled  in- 
stead of  smooth.  They  differed  from  sugary  seeds,  however,  in 
being  generally  less  wrinkled  and  having  a  more  shrunken  appear- 
ance. In  fact,  except  for  the  translucent  condition,  they  were  not 
unlike  the  shrunken  seeds  described  by  Hutchison. 

On  examination,  the  endosperm  of  these  aberrant  seeds  was 
found  to  consist  of  a  mass  of  amorphous  tissue,  distinctly  sweetish 
to  the  taste  and  very  brittle  in  texture.  Because  of  this  latter 
feature  the  new  character  has  been  given  the  name  brittle  endo- 
sperm and  the  factor  symbol  bt. 


*  Prof.  J.  B.  Wentz,  Iowa  State  College,  writes  that  he  has  recently  sent 
to  the  Journal  of  Heredity  a  manuscript  in  which  he  describes  this  same 
character  as  concave. 


580  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    2JO, 

Some  of  the  brittle  seeds  from  one  of  these  ears  were  planted 
in  1923.  Though  the  germination  was  poor  and  the  plants  lacked 
somewhat  in  vigor,  a  few  self-pollinated  pure  brittle  ears  were 
obtained.  The  original  segregating  ear  and  one  of  the  brittle 
ears  derived  from  it  are  shown  in  Figure  71. 

When  outcrossed  to  an  unrelated  stock  the  brittle  seeds  re- 
appeared in  the  F2  endosperm  generation  as  simple  Mendelian 
recessives.  A  count  of  the  normal  and  brittle  seeds  from  five  self- 
pollinated  ears  of  such  a  cross  is  given  in  Table  18. 

Table  18.     Starchy  and  Brittle  Seeds  on  Self-pollinated  Ears. 

Ear  No.  Starchy  Brittle 

1747  212  6l 

1748  349  91 


1749 

273 

93 

1750 

190 

57 

1751 

335 

104 

Total 

1359 

406 

Ex.  3:1 

1324 

441 

Deviation  35 

P.  E.,  12.27 

Dev./P.  E.,  2.9 

Because  this  new  character  resembled,  in  some  respects,  both 
sugary  and  shrunken  endosperm,  it  was  crossed  with  both  of 
these.  The  F1  seeds  in  both  series  of  crosses  were  starchy,  indi- 
cating that  brittle  endosperm  is  a  condition  genetically  different 
from  either  sugary  or  shrunken. 

SUGARY  X  BRITTLE 

Although  the  brittle  seeds  on  the  original  segregating  ear  were 
similar  to  sugary  seeds  in  appearance,  the  two  types  were  dis- 
tinguishable and  could  be  separated  with  a  fair  degree  of  accuracy 
when  both  occurred  on  the  same  ears.  In  addition  to  the  general 
differences  already  mentioned,  it  was  noted  that  on  ears  of  which 
all  the  seeds  were  genetically  white,  the  brittle  seeds  were  char- 
acterized by  a  faint  yellowish  cast,  a  sort  of  discoloration  of  the 
endosperm,  while  the  sugary  seeds,  like  the  starchy,  were  a  clear 
white. 

Table  19  gives  the  results  of  separating  the  starchy,  sugary, 
and  brittle  kernels  from  three  ears  and  Figure  72  illustrates  the 
P1?  Fx,  and  F2  ears  of  this  cross. 

Although  the  results  fit  a  9 13 :4  ratio  the  deviation  from 
expectation  is  rather  high,  there  being  an  excess  of  starchy  and  a 
deficiency  of  sugary  seeds  greater  than  four  times  the  probable 
error.  The  deviation  of  the  brittle  seeds  is  within  the  limits  of 
random  sampling.     The  marked  deviation  in  the  starchy  :  sugary 


ENDOSPERM    CHARACTERS    IN    MAIZE 


5*-i 


Fig.  71. — Original  ear  of  Sanford  White  segregating  for  brittle 
endosperm  and  a  pure  brittle  ear  derived  from  it. 


ratio  may  be  due  to  genetic  factors  affecting  the  rate  of  pollen 
tube  growth  which  are  discussed  in  Part  V,  published  elsewhere 


Sugary 

Brittle 

61 

47 
43 

78 
63 
72 

151 
176 

—25 

213 

235 

— 22 

582  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

( Mangelsdorf  and  Jones,  1926).  In  spite  of  these  deviations  the 
results  show  that  sugary  and  brittle  endosperm  are  due  to  two  dis- 
tinct genetic  factors  which  are  probably  independent  in  inheritance, 
no  indications  of  linkage  being  shown  in  this  cross. 

Table  19.     Starchy,  Sugary  and  Brittle  Seeds  in  the  F2  Endosperm  Gen- 
eration of  a  Cross  between  Sugary  and  Brittle. 

Ear  No.  Starchy 
422  242 

1754  169 

1755  166 

Total  577 

Ex.   9  :_3  -.4  530 

Deviation  47 

P.  E.  9:7  ratio  10.26 
Dev./P.  E.  4.6 

SHRUNKEN  X  BRITTLE 

Only  two  segregating  F2  progenies  of  this  cross  were  obtained. 
A  count  of  the  seeds  on  these  two  ears  is  shown  in  Table  20. 
Because  of  the  variation  in  the  translucent  condition  of  the 
brittle  seeds,  an  accurate  separation  of  brittle  and  shrunken  in- 
dividuals was  not  possible  and  these  two  classes  have  been 
combined. 

It  should  be  mentioned  that  both  of  these  ears  were  also  segre- 
gating for  sugary  seeds.  These  could  not  be  classified  accurately 
with  regard  to  the  other  characters  involved  in  this  cross  and 
since  sugary  is  known  to  be  independent  of  both  shrunken  and 
brittle  it  was  considered  safe  to  disregard  the  unclassified  sugary 
seeds  entirely. 

Table  20.     Starchy,    Shrunken,   and   Brittle   Seeds  in   the  F2  Endosperm 
Generation  of  a  Cross  between  Shrunken  and  Brittle. 

Shrunken  and  Brittle 
90 
6l 

151 
I7I-5 


Although  there  is  a  deviation  of  3.1  times  the  probable  error, 
the  results  approach  a  9.7  ratio.  This  indicates  that  brittle  and 
shrunken  endosperm  are  due  to  two  distinct  genetic  factors  which 
are  complementary  in  their  action  and  are  inherited  independently. 
Linkage  between  the  bt  and  sh  factors  would  be  indicated  by  an 


Ear  No. 

Starchy 

1756 
1757 

122 

119 

Total 
Ex.  9:7 
Deviation,  20.5 
P.  E.  6.62 
Deviation/P.  E.,  3.1 

241 
220.5 

ENDOSPERM    CHARACTERS    IN    MAIZE 


583 


Fig.  J2. — Parental,  Fi  and  F2  generations  of  a  cross  between  sugary 
and  brittle  endosperm. 

excess    of    the   recessive   types ;     actually   there   is    a    significant 
deficiency. 


BRITTLE  ENDOSPERM   FROM  TWO  VARIETIES 

In  1923,  Dr.  R.  A.  Emerson,  in  a  letter  to  Dr.  D.  F.  Jones, 
wrote  that  wrinkled,  translucent  seeds  had  been  found  in  an  inbred 


584  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

strain  of  Improved  Learning.  These  seeds  were  first  thought  to 
be  sugary  seeds  segregating  out  from  a  previous  accidental  cross. 
The  recessive  seeds,  however,  failed  to  germinate  in  an  incubator 
and  heterozygous  plants  pollinated  by  pure  sugary  gave  only 
normal  starchy  seeds.  These  facts  led  to  the  conclusion  that  the 
new  character  was  a  lethal,  phenotypically  similar  to  sugary  but 
genetically  quite  distinct  from  the  latter. 

Dr.  Emerson  very  kindly  sent  seed  of  the  original  strain  and  of 
the  cross.  Both  lots  were  grown  in  1923  and  a  number  of  self- 
pollinated  ears  were  obtained.  Though  the  recessive  seeds  of  the 
original  inbred  strain  failed  to  germinate,  the  extracted  recessives 
from  the  vigorous  F1  ears  grew  readily.  When  pollen  from  these 
pure  recessives  of  Emerson's  strain  was  applied  to  plants  hetero- 
zygous for  the  brittle  factor,  a  1:1  segregation  of  starchy  and 
brittle  occurred,  demonstrating  that  this  new  endosperm  character 
from  Improved  Learning  is  genetically  identical  to  the  brittle 
endosperm  found  in  Sanford  White.  Improved  Learning  is  a 
dent  variety  of  Western  origin  while  Sanford  White  is  a  New 
England  Flint  type  of  long  standing. 

SHRUNKEN  ENDOSPERM   FROM    TWO   SOURCES 

In  this  connection  it  may  be  of  interest  to  record  that  the  char- 
acter shrunken  which  Hutchison  found  in  some  ears  of  floury 
corn  from  the  Ponka  Indian  reservation  has  also  appeared  in  a 
Kansas  variety  of  dent  corn  known  as  Pride  of  Saline.  Open 
pollinated  ears  with  a  few  shrunken  seeds  were  sent  to  the  writer 
by  Professor  J.  H.  Parker  of  the  Kansas  Experiment  Station. 
The  recessive  seeds  from  these  ears  were  grown  and  crosses  were 
made  between  this  strain  and  a  shrunken  stock  secured  from  Dr. 
Hutchison.  The  F1  seeds  of  such  crosses  were  all  pure  shrunken, 
demonstrating  the  genetic  similarity  of  the  two  parental  types 
with  respect  to  this  character. 

WAXY  ENDOSPERM  IN  CHINA  AND  AMERICA 

Of  even  greater  interest  is  the  discovery  in  1922  of  waxy  endo- 
sperm in  two  self-pollinated  ears  of  Sanford  White,  though  of  a 
different  strain  from  the  one  in  which  brittle  endosperm  was 
found. 

This  peculiar  endosperm  texture  had  previously  been  found 
only  in  three  isolated  localities  in  China,  Burma,  and  the  Philip- 
pines. Collins  (1920)  failed  to  find  a  single  waxy  variety  among 
more  than  a  thousand  American  varieties  which  he  examined. 

The  waxy  type  isolated  from  Sanford  White  was  crossed  with 
a  waxy  strain  which  had  come  originally  from  Shanghai,  China. 
This  cross  gave  only  pure  waxy  Fx  seeds,  proving  that  the  two 
strains  were  genetically  alike  in  their  endosperm  texture. 


ENDOSPERM    CHARACTERS    IN    MAIZE  585 

So  far  as  is  known,  the  only  waxy  corn  ever  grown  in  Con- 
necticut is  the  Chinese  strain  which  has  been  used  in  genetic 
investigation  at  the  experiment  station  farm  for  a  number  of 
years.  No  corn  of  any  kind  has  ever  been  sent  from  the  station 
to  the  locality  from  which  the  ears  of  Sanford  White  were 
obtained,  and  it  is  scarcely  possible  that  the  appearance  of  waxy 
endosperm  in  this  variety  is  due  to  previous  crossing  with  the 
Chinese  waxy.  Nor  is  there  any  indication  that  the  strain  in 
which  waxy  has  appeared  has  undergone  any  recent  crossing  with 
such  a  widely  different  sort  as  the  Chinese  waxy.  The  strain 
which  carries  the  waxy  endosperm  is  typical  of  the  variety  in 
every  respect,  including  number  of  rows  of  grain  on  the  ear  which 
Collins  (in  a  letter)  suggests  as  the  character  which  would  be 
most  affected  by  crossing. 

The  origin  in  an  American  variety  of  this  peculiar  endosperm 
texture,  previously  found  only  in  several  isolated  Asiatic  localities, 
will  probably  remain  a  matter  for  speculation.  It  may  have 
arisen  as  a  mutation  in  very  recent  years  or  it  may  have  been 
carried  by  the  stock  as  a  hidden  recessive  for  centuries.  Waxy 
seeds  are  not  particularly  conspicuous  in  appearance  and  a  few 
such  seeds  on  open  pollinated  ears  would  ordinarily  escape  atten- 
tion, and  the  character  might  be  carried  along  indefinitely. 
Neither  is  a  recent  mutation  from  starchy  to  waxy  an  impossi- 
bility, since  mutations  affecting  the  endosperm  have  appeared  in 
homozygous  inbred  strains  of  maize  in  at  least  four  instances,  as 
noted  previously. 

CONSTANT  VARIATION  IN  THE  STORAGE  MATERIAL  OF 
THE  ENDOSPERM 

The  finding  of  brittle  endosperm  in  Learning  and  Sanford 
White,  of  shrunken  endosperm  in  Pride  of  Saline  and  a  variety 
of  flour  corn  from  the  Ponka  Indians,  of  waxy  endosperm  in 
China,  Burma,  the  Philippines,  and  finally  in  New  England,  leads 
to  the  conclusion  that  these  recessive  characters  have  a  wide  dis- 
tribution in  the  germplasm  of  maize  varieties,  or  that  the  germ- 
plasm  is  constantly  producing  anew  these  variations  which  affect, 
so  profoundly,  the  nature  of  the  food  material  stored  in  the  endo- 
sperm. Under  domestication  some  of  these  variations,  as  for 
example,  sugary,  offer  economic  advantages  and  are  retained. 
Others  may  have  a  survival  value  under  certain  climatic  condi- 
tions and  are  automatically  sorted  out.  This  may  have  been  the 
origin  of  waxy  varieties  in  China,  Burma,  and  the  Philippines. 

It  is  now  generally  agreed  that  maize  was  confined  to  the 
American  continent  previous  to  the  beginning  of  the  sixteenth 
century.  Why  waxy  varieties  should  have  been  developed  in 
these  Asiatic  localities  and  apparently  in  no  other  part  of   the 


586  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

world  is  difficult  to  understand,  unless  this  peculiar  type  of  endo- 
sperm has  natural  advantages  under  the  environmental  conditions 
obtaining  in  these  regions,  or  unless  waxy  varieties  were  isolated 
because  of  their  economic  superiority.  Since  the  waxy  corn  is  gen- 
erally regarded  as  inferior  to  the  starchy  types  for  food  purposes, 
where  both  are  known,  it  seems  more  likely  that  the  isolation  of 
waxy  varieties  has  been  brought  about  by  natural  selection.  This 
assumption  is  substantiated  by  the  discovery  of  waxy  endosperm 
in  varieties  of  Coix  and  Andropogon  sorghum  from  these  same 
regions.      (Kempton  1921.) 


THE  RELATIVE  DEVELOPMENT  OF  ENDOSPERM  CHARACTERS 

The  fact  that  the  brittle  seeds  show  such  a  low  percentage  of 
germination,  in  some  cases  none  at  all,  suggests  that  these  various 
factors  which  affect  the  texture  of  the  endosperm  are  not  funda- 
mentally different  from  the  lethal  and  semi-lethal  seeds  described 
in  Part  I ;  that  all  represent  "defective"  conditions  of  the  endo- 
sperm, and  that  any  distinction  which  is  made  is  one  of  degree 
rather  than  of  kind. 

It  will  be  recalled  that  when  both  normal  and  defective  seed 
from  segregating  ears  were  weighed,  the  defectives  showed  a 
development  ranging  from  2.4  per  cent  in  the  completely  lethal 
types  to  59  per  cent  in  the  semi-lethal  types. 

The  same  method  of  determining  the  relative  development  of 
these  five  endosperm  characters  has  been  followed.  The  domi- 
nant and  recessive  seeds  from  segregating  ears  were  separated, 
counted,  and  weighed.  The  average  weight  of  each  type  was 
determined  by  simple  division  and  the  relative  development 
obtained  by  a  comparison  of  the  two  quotients. 

The  average  weights  of  the  seeds  and  the  relative  development 
of  each  type  of  endosperm  texture  is  shown  in  Table  21. 


Table  21.     Average   and    Relative   Weights   of    Dominant   and   Recessive 
Seeds  fom  Segregating  Ears. 

Character  No.  of         Av.  Wt.  Dominant    Av.  Wt.  Recessive       Rel.  Wt.  of 


Segregating 

Ears 

Seeds  in  mg. 

Seeds  in  mg. 

Recessives 

Waxy- 

3 

306 

295 

96.5 

Floury 

3 

208 

199 

95-6 

Shrunken 

3 

321 

295 

91.9 

Sugary 

5 

255 

226 

88.5 

Brittle 

2 

230 

143 

62.3 

It  will  be  noted  that  in  evesy  case  the  recessive  seeds  were  lower 
in  weight  than  the  dominant  seeds  from  the  same  ears.  Hutchison 
(1921 )  weighed  a  large  number  of  individual  starchy  and  shrunken 
seeds    from    segregating   ears    and   though    the    shrunken    seeds 


ENDOSPERM    CHARACTERS    IN    MAIZE  587 

were  lower  in  weight  the  difference  was  not  significant  when  the 
probable  error  was  considered.  He  concluded,  therefore,  that  the 
shrunken  seeds  were  equal  to  the  starchy  seeds  in  their  develop- 
ment. Although  the  method  used  here  does  not  permit  the  calcu- 
lation of  probable  errors  it  does  give  a  very  accurate  average 
weight  of  the  dominant  and  recessive  seeds.  Of  the  many  segre- 
gating ears  which  have  been  used  in  these  and.  other  determina- 
tions not  one  has  ever  been  found  in  which  the  recessive  seeds 
were  equal  in  weight  to  the  dominant  ones. 

Apparently  the  seed  of  maize  attains  its  maximum  development 
only  when  the  endosperm  is  genetically  starchy.  Genetic  factors 
which  cause  the  formation  of  other  carbohydrates  in  the  endo- 
sperm, such  as  dextrose  in  the  case  of  sweet  corn,  possibly  some 
other  sugar  in  the  brittle  seeds,  and  perhaps  erythrodextrin  in  the 
waxy  seeds  (Weatherwax  1922),  do  so  at  the  expense  of  total 
dry  matter  laid  down.  Although  the  amount  of  dry  matter  is  not 
necessarily  the  sole  criterion  of  endosperm  achievement,  it  never- 
theless appears  to  be  the  most  important  one. 

The  waxy  seeds  most  nearly  approach  the  normal  condition  in 
relative  development,  the  floury  seeds  come  next,  followed  by 
shrunken,  sugary  and  brittle  in  the  order  given.  The  germina- 
tion of  the  seeds  and  the  vigor  of  the  seedlings  is  closely  correlated 
with  the  relative  development  of  each  type,  the  waxy  and  floury 
seeds  being  almost  equal  to  starchy  in  these  respects  while  the 
shrunken  and  sugary  seeds  are  somewhat  inferior  and  the  brittle 
seeds  are  very  poor. 

DISCUSSION 

These  five  characters  which  influence  the  texture  of  the  endo- 
sperm and  the  thirteen  factors  which  govern  the  amount  of  tissue 
can  be  arranged  in  a  series  representing  different  stages  of  endo- 
sperm formation.  All  of  the  factors  result  in  a  decreased  develop- 
ment of  the  endosperm  as  measured  by  the  dry  weight  of  the 
seed.  When  the  relative  development  of  these  six  types  is  repre- 
sented by  points  on  the  normal  growth  curve  of  the  seed  of  maize, 
together  with  the  thirteen  defectives,  as  shown  in  Figure  73,  it 
is  noted  that  these  18  endosperm  characters  form  a  continuous 
series  ranging  from  the  de14  type  to  waxy  endosperm. 

In  other  words,  the  seeds  of  maize,  to  attain  a  relative  develop- 
ment of  100  per  cent,  must  pass  safely  through  all  the  points 
represented  on  the  growth  curve.  This  means  that  in  order  to 
attain  the  normal  or  "starchy"  condition  a  seed  must  be  dominant 
for  at  least  18  genetic  factors,  any  one  of  which  in  the  homozy- 
gous recessive  condition  would  reduce  its  development. 

The  space  on  the  curve  between  50  and  90  is  not  heavily  popu- 
lated and  it  is  possible  that  characters  which  fall  into  this  region 
are  still  to  be  found. 


588  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 


FERTILIZATION 


Fig.  73. — Diagram  in  which  the  relative  development  of  endosperm 
characters  is  represented  by  points  on  the  growth  curve  of  normal  seeds 
of  maize. 


The  characters  which  show  a  development  of  50  per  cent  or 
more  are  the  most  useful  to  the  geneticist  because  there  is  enough 
endosperm  development  for  differences  in  texture  to  be  apparent 


ENDOSPERM    CHARACTERS    IN    MAIZE  589 

and  in  most  cases  these  characters  can  be  secured  in  a  homozy- 
gous condition.  This  latter  feature  constitutes  an  advantage 
when  linkage  relations  are  studied  because  it  permits  the  use  of 
backcrosses. 

Summary — Part  III 

1.  Brittle  endosperm,  a  new  character  intermediate  between 
sugary  and  shrunken  in  appearance,  is  inherited  as  a  simple 
recessive. 

2.  Crosses  between  brittle  and  sugary  or  brittle  and  shrunken 
give  starchy  seeds  in  Ft  and  9 :3  4  or  9  :y  ratios  in  F2.  No  indi- 
cation of  linkage  is  shown  in' either  series  of  crosses. 

3.  Brittle,  shrunken,  and  waxy  endosperm  have  been  found 
in  widely  separated  localities  and  in  unrelated  varieties  indicating 
their  widespread  distribution  in  the  germplasm  or  constant 
reappearance  by  mutation. 

4.  The  endosperm  of  maize  apparently  attains  its  maximum 
development  when  genetically  starchy.  The  relative  development 
of  waxy,  floury,  shrunken,  sugary  and  brittle  seeds  is  always  less 
than  that  of  starchy  seeds  from  the  same  ears. 

5.  The  characters  which  affect  the  texture  of  the  endosperm 
are  not  fundamentally  different  from  the  defective  seeds  pre- 
viously described,  which  influence  primarily  the  amount. 

6.  The  maize  seed  in  order  to  attain  the  normal  or  "starchy" 
condition  must  be  dominant  for  at  least  18  2'enetic  factors. 


59°  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 


PART    IV 

Premature  Germination  of  Maize  Seeds  and  Genetic 
Factors  Which  Govern  Dormancy 

In  a  paper  on  defective  seeds,  the  writer  (1923)  mentioned 
briefly  a  condition  in  maize  in  which  the  seeds  fail  to  go  through 
a  resting  period,  germinating  while  still  attached  to  the  ear  of  the 
growing  plant  and  before  the  seed  is  mature.  A  similar  condition 
was  reported  simultaneously  by  Lindstrom  (1923)  who  found 
among  the  ears  of  a  strain  of  Golden  Bantam  sweet  corn,  which 
was  being  studied  for  defective  endosperm,  one  ear  which  also 
segregated  for  germinating  seeds.  Somewhat  later  Eyster  (1924) 
described  a  similar  character  which  he  calls  "primitive  sporo- 
phyte,"* and  more  recently,  in  another  paper  (Eyster,  1924),  he 
reports  a  second  factor  for  premature  germination. 

Since  1921  a  number  of  stocks  which  segregate  for  germinating 
seeds  have  come  under  observation  of  the  writer.  Self-pollina- 
tions and  crosses  in  these  stocks  indicate  that  there  are  at  least  five 
complementary  factors  and  two  sets  of  duplicate  factors  involved 
in  premature  germination  of  maize  seeds.  A  description  of  these 
characters  and  an  account  of  their  genetic  behavior  is  given  in 
the  following  pages. 

COMPLEMENTARY  FACTORS 

The  first  type  of  premature  germination  was  found  in  1921  in 
a  strain  of  Gold  Nugget  flint  corn  which  had  been  twice  self- 
pollinated.  The  germinating  seeds  were  not  noted  in  the  first 
generation  and  either  had  not  appeared,  or  the  germination  was  so 
slight  as  to  escape  notice.  The  ratio  of  dormant  to  germinating 
seeds  was  approximately  3:1  and  the  character  has  continued  to 
behave  as  a  simple  Mendelian  recessive  both  in  the  original  strain 
and  in  crosses.  A  count  of  the  dormant  and  germinating  seeds 
from  eight  ears  of  this  stock  is  shown  in  Table  22. 

Premature  germination  induced  by  the  ge1  factor  begins  at  an 


*  The  term  primitive  sporophyte  as  used  by  Eyster  in  describing  pre- 
mature germination  in  maize,  has  not  been  adopted  by  the  writer  because 
it  is  thought  to  be  somewhat  misleading.  Eyster  compares  the  condition 
in  maize  to  that  found  in  Selaginella  ruprestris  in  which  the  sporophyte 
develops  while  the  egg  is  still  attached  to  the  female  gameteophyte.  The 
resemblance  between  this  condition  and  that  found  in  maize  is  more  super- 
ficial than  real.  As  a  mere  term  to  designate  the  character,  primitive 
sporophyte  would  serve  as  well  as  any,  except  for  the  fact  that  readers, 
not  familiar  with  the  premature  germination  in  maize,  might  draw  the 
conclusion  that  prematurely  germinating  seeds  actually  represent  a  rever- 
sion to  the  primitive  condition  of  the  race  and  that  a  change  in  a  single 
gene  can  carry  back  the  maize  plant,  phylogenetically,  so  to  speak,  to  the 
group  in  which  the  club  mosses  are  found. 


ENDOSPERM    CHARACTERS    IN    MAIZE 


59* 


early  stage.     The  actual  sprouting-  of  the  seed  has  usually  occurred 
when  the  seeds  are  in  the  late  milk  stage  but  the  segregation  is 


Fig.  74. — 'Two  ears  segregating  for  germinating  seeds  of  the  gei 
type.  Germination  begins  at  an  early  stage.  The  ear  at  the  right 
shows  the  apparent  linkage  between  endosperm  color  and  premature 
germination. 

apparent  at  an  earlier  period  because  the  recessive  seeds  are,  with 
few  exceptions,  white,  while  the  dormant  seeds  on  the  same  ear 
are  yellow.     Eyster   reports   a   similar  association   between  pre- 


592  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    2/9 

mature  germination  and  the  absence  of  color  in  the  endosperm  and 
explains  this  on  the  basis  of  close  linkage.  Evidence  presented 
later  suggests  that  this  may  not  be  the  true  explanation,  but  the  fact 
remains  that  the  germinating  seeds  of  this  type  are  almost  always 
lacking  in  endosperm  color  at  all  stages  and  can  be  distinguished 
from  the  dormant  seeds  as  soon  as  the  latter  begin  to  show  color. 
When  the  seeds  have  reached  the  late  milk  stage,  germination  has 
proceeded  to  a  marked  degree.  The  plumule,  though  well  devel- 
oped, is  often  unable  to  burst  the  pericarp  because  the  endosperm 
is  still  soft  and  no  pressure  is  provided.  Instead  it  winds  aim- 
lessly around  the  endosperm,  giving  the  seed  a  peculiarly  swollen 
appearance.  Later,  as  the  endosperm  tissues  harden,  many  of  the 
plumules  are  able  to  rupture  the  seed  coat  and,  often,  well 
developed  roots  and  shoots  are  formed. 

Germinating  seeds,  removed  from  the  ear  and  grown  in  sand 
in  the  greenhouse,  produce  only  albino  seedlings  which  are 
very  abnormal  in  appearance  and  soon  die.  If  such  seedlings  are 
grown  in  complete  darkness  they  develop  a  faint  green  color  which 
disappears  immediately  upon  exposure  to  full  light.  The  faint 
green  color  is  also  formed  when  the  seeds  remain  on  the  ear, 
well  covered  by  heavy  husks,  but  disappears  when  the  husks  are 
stripped  down. 

Premature  germination  of  the  type  caused  by  the  ge2  factor 
was  found  in  an  inbed  strain  of  Golden  Bantam  sweet  corn.  This 
type  of  germination  is  not  nearly  so  complete  as  that  caused  by 
the  ge1  factor.  The  recessive  seeds  are  recognized  by  their  smaller 
size  and  pale  yellow  endosperm,  but  not  all  of  them  show  well 
marked  indications  of  sprouting,  and  only  occasionally  is  germina- 
tion so  advanced  that  the  plumule  ruptures  the  pericarp,  and  pri- 
mary or  secondary  roots  are  formed.  When  these  occasional 
seeds  which  do  show  marked  germination  are  removed  from  the 
ear  and  grown  in  the  greenhouse,  they  develop  normally  so  far 
as  chlorophyll  is  concerned,  but  are  considerably  dwarfed  in 
appearance,  growing  more  slowly  than  normal  seedlings.  Only 
a  few  of  them  survive  more  than  several  weeks. 

The  condition  caused  by  the  ge2  factor  is  also  inherited  as 
a  simple  recessive  and  a  count  of  seven  ears  segregating  for  this 
character  is  given  in  Table  22. 


Table  22. 

Seg 

regation 

of  Dormant  and 

Germim 

tin 

g  Seed 

5  in  Stoc 

ge%  and 

gd. 

Type 

No 

of  Ears 

Total 

ge 

Ex.  3:1 

Dev. 

P.  E. 

gei 

8 

2220 

S28 

555 

—27 

13.8 

gC2 

7 

I928 

509 

482 

• 

27 

12.8 

ge3 

4 

446 

99 

112 

—13 

6.2 

get 

1 

169 

44 

42 

2 

3.8 

ENDOSPERM    CHARACTERS    IN    MAIZE  593 

In  1924,  Dr.  Lindstrom  very  kindly  furnished  the  writer  with 
seed  of  his  strain  which  had  heen  segregating  for  germinating 
seeds.  In  some  respects  this  type  of  premature  germination, 
designated  as  ge3  in  this  series,  is  almost  identical  to  ger.  The 
germinating  seeds  are  white,  with  few  exceptions,  while  the 
dormant  seeds  have  a  deep  yellow  color  so  characteristic  of  the 
Golden  Bantam  variety.  The  plumules  produced  by  the  germi- 
nating seeds  are,  like  those  of  get,  albinos.  They  differ  from 
the  latter,  however,  in  failing  to  form  the  faint  tinge  of  green 
which  characterizes  the  ge1  seedlings  under  certain  conditions. 

The  processes  of  germination  in  the  ge3  seeds  evidently  begin 
earlier  and  proceed  further  than  in  gex  seeds.  When  the  dor- 
mant seeds  are  mature,  the  endosperm  of  the  germinating  seeds 
is  almost  completely  exhausted  and  the  pericarp  is  little  more  than 
an  empty  shell  containing  the  withered  remains  of  the  partly 
grown  seedling.  This  character  is  inherited  as  a  simple  reces- 
sive as  indicated  by  the  counts  of  the  seeds  from  four  ears  shown 
in  Table  22. 

Germinating  seeds  were  noted  on  an  ear  of  an  eight-rowed, 
yellow  flint  variety  of  the  Longfellow  type,  received  from  Mr. 
T.  B.  Macaulay  of  Montreal,  Canada.  No  count  of  the  seeds  was 
made  on  the  original  ear  which  was  thought  to  be  open-pollinated. 
When  the  dormant  seeds  from  this  ear  were  grown  and  the  plants 
self-pollinated,  segregating  ears  were  again  obtained.  The  pro- 
portion of  dormant  to  germinating  seeds  on  one  of  these  ears  is 
given  in  Table  22. 

Germination  in  this  stock  does  not  begin  until  the  seeds  are 
fairly  hard.  The  premature  germination  is  not  associated  with 
absence  of  color  in  the  endosperm  as  in  the  three  stocks  already 
described,  nor  are  the  seedlings  albinos  as  in  the  case  of  gex  and 
g"£3.  Germinating  seeds  of  this  type,  when  removed  from  the 
ear  and  planted  in  the  greenhouse,  produce  seedlings  which  are 
almost  normal  in  appearance.  By  growing  a  large  number  of 
such  seedlings  it  might  be  possible  to  mature  a  few  homozygous 
plants  in  the  greenhouse  during  the  winter  months.  This  stock, 
however,  is  the  only  one  in  which  there  seems  to  be  any  possi- 
bility of  ever  attaining  a  homozygous  condition. 

This  factor  evidently  arose  as  a  mutation  in  either  the  ge1  or 
£■£3  stocks.  A  cross  between  these  two  stocks,  in  addition  to  giving 
the  expected  9:7  and  3:1  ratios,  also  produced  a  few  progenies 
in  which  the  ratio  was  clearly  27  137,  as  the  figures  in  Table  24 


594  CONNECTICUT   EXPERIMENT   STATION  BULLETIN    279 

indicate.  Evidently  three  factors  for  germinating-  seeds  instead 
of  two  were  involved  in  this  cross.  It  is  not  known  which  of 
the  parental  stocks  contributed  the  third  factor  but  it  seems  fairly 
certain  that  it  arose  in  one  or  the  other  by  recent  mutation,  since 
both  strains  had  previously  given  only  mono-hybrid  ratios  and  a 
later  generation  has  done  the  same. 

This  new  factor  is  almost  identical  to  ge3  in  its  effect.  The 
recessive  seeds  are  completely  lacking  in  endosperm  color  and  the 
sprouts  are  entirely  without  chlorophyll.  The  two  types  cannot 
be  distinguished  from  each  other  but  both  are  separable  from  gex 
for  a  brief  period  during  which  the  faint  green  color  is  visible  in 
the  sprouts  of  the  latter. 

DESCRIPTION   SUMMARIZED 

The  chief  characteristics  of  these  five  types  of  germinating  seeds 
may  be  briefly  summarized  as  follows : 


J 

approximate   Time 

Germination 

Color  of 

Color  of 

Type 

Begins 

Endosperm 

Plumule 

gei 

late  milk 

white 

white  (green  tinge) 

get 

dough 

pale  yellow 

green 

ge3 

early  milk 

white 

white 

gei 

hard  dough 

yellow 

green 

get 

early  milk 

white 

white 

It  should  be  emphasized  that  all  these  stocks  are  genetically 
yellow  in  their  factors  for  endosperm  color  and  genetically  green 
with  regard  to  seedling  color.  The  absence  of  endosperm  and 
chlorophyll  color  is  apparently  due  to  the  physiological  effects  of 
premature  germination. 

All  these  characters  are  fully  as  lethal  in  their  effect  as  the 
defective  seeds  described  in  Part  I.  Under  field  conditions  the 
growing  seedling  dies  as  soon  as  the  plant  matures  and  the  mois- 
ture supply  is  cut  off. 

PHENOTYPICAL  AND  GENETIC  DIFFERENCES 

The  breeding  program  with  these  five  types  of  premature  ger- 
mination involves  crossing  them  in  all  combinations.  Only  seven 
of  the  ten  possible  crosses  have  so  far  been  made,  but  the  dis- 
tinct phenotypical  differences  between  several  of  the  types  almost 
precludes  the  possibility  that  they  are  genetically  alike,  and  a 
tentative  conclusion  has  been  reached  that  all  five  are  probably 
genetically  distinct. 

It  is  noted  that  the  types  fall  into  two  distinct  classes  with 
regard  to  plumule  color,  ge1}  ge3,  and  ges  producing  white  sprouts, 
ge2  and  ge±  producing  only  green  plumules.     That  the  first  three 


ENDOSPERM    CHARACTERS    IN    MAIZE 


595 


are  genetically  distinct  is  shown  by  the  2.7  :$J  ratios  given  in 
Table  24.  The  ge2  and  ge±  types  are  considered  to  be  distinct 
from  the  remaining  three  because  of  the  marked  phenotypical 
differences  between  the  two  groups,  and  crosses  which  have  so  far 
been  made  confirm  this  assumption.  The  cross  between  ge2  and 
ge±  has  not  yet  been  made  and  though  these  two  differ  in  the 
amount  of  endosperm  color,  the  time  at  which  germination  begins, 
and  the  vigor  of  the  seedlings,  these  differences  are  all  of  degree 
and  might  result  from  the  action  of  modifying  factors.     There 


ges 


Seeds  Normal 


Two   Types  in  F2 
Di-hybrid  Ears   in  F2 
Phenotypical  Differences 


Fig.  75. — Diagram  showing  the  crosses  which  have  been  made  among 
the  five  stocks  in  which  complimentary  factors  governing  premature 
germination  are  involved. 

remains  the  possibility,  therefore,  that  ge2  and  ge±  are  genetically 
identical. 

The  situation  with  regard  to  these  five  types  is  shown  in  Figure 
75.  Squares  with  vertical  cross  hatching  represent  crosses  in 
which  the  F1  seeds  are  dormant  and  the  F2  has  not  yet  been 
grown.  Those  with  horizontal  cross  hatching  indicate  the  com- 
binations in  which  both  types  were  recovered  in  F2  although  no 
di-hybrid  ears  were  obtained.  Diagonal  cross  hatching  repre- 
sents crosses  in  which  di-hybrid  ratios  were  found  in_F2,  while 
two  intersecting  diagonal  lines  indicate  marked  phenotypical  differ- 


596 


CONNECTICUT    EXPERIMENT    STATION 


BULLETIN    2/9 


ences  between  the  types.     The  crosses  in  which   di-hybrid  ears 
were  obtained  are  given  in  detail  in  Tables  23  and  24. 

Table  22.     Ears  Segregating  for  Both  Factors  in  the  E2  Endosperm  Gen- 
eration of  a  Cross  of  gei  and  gei. 
Ear  No.  Dormant  Germinating 

711  149  99 

712  139  112 

713  146  118 

714  168  130 


Total 
Ex.  9:7 
Dev.  5 
P.  E.  10.9 


602 

597 


459 
464 


Table  24.     Mendelian  Ratios  Occurring  in  the  F?  Endosperm  Generation 
of  a  Cross  Involving  gei,  gez  and  geo. 

Ear  No.  Dormant         Germinating-         Types  Involved     Ratio  Expected 

2037  172  57  gei  3:1 

2038  230  78  gei 
2040               216                 22  gei 


Total 
Ex.  3:1 


618 
582 


157 
194 


2039 
2041 
2042 
2043 
2044 
2045 
2046 

2047 

Total 
Ex.  9:7 


122 

165 
190 

131 
212 

184 

128 
171 

1303 
1259 


105 
124 
129 

83 
127 
126 

113 
T28 

935 
979 


gez,  ge-o 

gez,  ge-o 
gei  and  gez  or  ge5 

gez,  ge-o 

gez,  ge-o 
gei  and  gez  or  ge-o 

gez,  ge-o 

gez,  geo 


9-7 


2048 
2049 
2050 

Total 
Ex.  27 :37 


91 

137 
94 

322 
341 


178 
184 
125 

487 


gei,  gez,  geo 
gei,  gez,  ge5 
gei,  gez,  ges 


27:37 


The  evidence  so  far  as  it  goes  indicates  that  these  five  types  are 
all  different  and  that  the  plant  must  be  homozygous  for  the  domi- 
nant allelomorphs  of  all  five  factors  in  order  for  all  of  its  seeds 
to  remain  dormant  until  maturity. 


DUPLICATE  FACTORS 


Premature  germination  due  to  the  action  of  duplicate  factors 
was  first  found  on  the  Fx  ears  of  a  cross  between  two  strains  of 


ENDOSPERM    CHARACTERS    IN    MAIZE  597 

Canada  Flint  which  had  been  inbred  for  four  generations.  Ger- 
minating seeds  had  never  been  noted  in  either  of  these  inbred 
strains  nor  did  they  appear  among  the  F1  crossed  seeds.  When 
the  Fj  plants  were  grown,  however,  a  number  of  the  ears  bore 
germinating  seeds,  scattered  at  random  over  the  inflorescence. 
These  ears  were  open-pollinated,  and  no  attempt  was  made  to 
determine  the  ratio  in  which  the  segregation  occurred.  Dormant 
seeds  from  one  of  these  ears  were  grown  and  five  self-pollinated 
ears  were  obtained.  Three  of  these  were  clearly  segregating  in  a 
3  :i  fashion.  The  fourth  deviated  from  a  3  :i  ratio  by  an  amount 
equal  to  ten  times  the  probable  error,  but  exactly  fitted  a  15:1 
ratio.     The  counts  on  these  ears  are  arranged  in  Table  25. 

Table  25.     Segregation  of  Dormant  and  Germinating  Seeds  when  Dupli- 
cate Factors  gee  and  ge-  are  Involved. 


Nearest 

Deviation  from 

iar  No. 

Dormant 

G. 

:rminating 

Ratio 

Mendelian 

Ratio 

Nearest  Ratio 

2171 

123 

46 

2.7:1 

3:i 

1.1    times    P.  E. 

2172 

104 

31 

3-4:l 

3:i 

.9 

2173 

121 

43 

2.8:i 

3:i 

•5        " 

2174 

230 

15 

I5-3:I 

15:1 

.1        " 

The  appearance  of  germinating  seeds  in  the  second  generation 
of  a  cross  between  strains  which  had  never  previously  shown 
these  characters  and  the  3  :i  and  15:1  ratios  obtained  in  F3  can  be 
explained  by  assuming  a  pair  of  duplicate  factors,  one  of  which 
was  contributed  by  each  of  the  inbred  strains.  These  factors 
are  given  the  symbols  ge6  and  ge7  and  the  following  genetic  con- 
stitution of  the  parents  and  Fx  plants  is  suggested : 

Parents  Ft  Plants 

Ge6Ge6ge7ge7  Geege6Ge7ge7 

ge6ge6Ge7Ge7 

The  F1  plants  when  selfed  should  give  15:1  ratios.  Whether 
or  not  they  did  could  not  be  determined  because  the  ears  were 
all  wind-pollinated.  The  dormant  seeds  from  these  plants  should, 
when  grown,  give  three  types  of  progenies  ;  ( 1 ) ,  non  segregating, 
(2),  segregating  3:1,  (3),  segregating  15:1.  Had  the  Ft  plants 
been  selfed  these  three  types  of  progenies  would  be  expected  in 
the  ratio  of  7  '.4 14  respectively.  The  non-segregating  ears  would 
be  expected  when  one  or  both  of  the  recessive  factors  is  lacking: 
3:1  ratios  should  occur  when  the  plant  is  homozygous  for  one 
and  heterozygous  for  the  other,  while  the  15  :i  ratios  are  expected 
on  plants  which  are  heterozygous  for  both  factors. 

It  is  noted  that  all  three  types  of  progenies  have  been  obtained 
although  the  number  of  selfed  ears  examined  is  too  small  to  deter- 
mine in  what  proportions  the  various  types  are  appearing. 


5 98  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

Apparently  the  two  factors  involved  in  this  stock  are  'indepen- 
dent. Linkage  would  be  indicated  by  a  distortion  of  the  15:1 
ratio,  an  excess  of  recessives  being  expected  in  the  coupling  phase, 
a  deficiency  in  the  repulsion  phase. 

Premature  germination  in  this  stock  is  somewhat  similar  to  that 
caused  by  ge2.  The  plumules  are  green  but  the  endosperm  color, 
instead  of  being  pale  yellow  throughout,  is  affected  only  in  the 
regions  adjacent  to  the  embryo.  The  endosperm  color  of  the 
dorsal  sides  of  the  seed  is,  in  most  cases,  full  yellow. 

ratios  of  8  :i  and  41  :i 

Another  stock  in  which  the  action  of  duplicate,  or  perhaps 
triplicate,  factors  is  suggested  is  the  result  of  pollinating  a  chimera 
plant  which  appeared  in  the  third  generation  of  inbreeding  in  a 
strain  of  Sanford  White,  with  pollen  from  an  unknown  yellow 
stock.  The  chimera  had  produced  a  pure  white  ear  shoot  and  it 
was  desired  to  obtain  both  selfed  and  crossed  seeds  from  this 
plant.  Accordingly  pollen  from  an  Ft  hybrid  of  two  inbred 
strains  of  yellow  flint  corn  was  applied,  but  unfortunately  no 
record  was  made  of  the  row  from  which  the  pollen  was  taken 
and  it  is  not  known  whether  this  particular  hybrid  had  ever 
produced  germinating  seeds.  It  is  known,  however,  that  the  in- 
bred strain  of  Sanford  White  in  which  the  chimera  appeared  had 
never  shown  germinating  seeds. 

Table  26.     Segregation  of  Dormant  and  Germinating  Seeds  when  Dupli- 
cate Factors  ges  and  gea  are  Involved. 

Nearest  Deviation  from 

Ear  No.      Dormant  Germinating-      Ratio        Mendelian  Ratio  Nearest  Ratio 

2178  165  4  41.3:1  15:1  3-i    times    P.  E. 

2179  163  22  7.4:1  15:1  4.6       " 

2180  187  21  8.9:1  15:1  3-3       " 

Four  self -pollinated  ears  were  obtained  from  the  yellow  seeds 
produced  by  cross-pollination  with  the  unknown  pollen  parent. 
Three  of  these  segregated  for  germinating  seeds  and  the  counts 
on  these  ears  are  set  forth  in  Table  26.  Two  of  the  ears  gave 
ratios  approximately  alike,  the  proportion  of  dormant  to  ger- 
minating seeds  being  about  8:1.  This  ratio  deviates  from  3:1  by 
more  than  nine  times  the  probable  error  and  from  15:1  by  5.6 
times  the  error.  The  third  ear,  No.  2178,  gave  a  ratio  of  41  :i 
which  differs  from  a  15:1  by  3.1  times  the  probable  error  and 
from  63 : 1  by  an  amount  equal  to  3.2  times  the  error. 

These  results  may  be  explained  on  the  basis  of  linkage  between 
the  two  members  of  a  pair  of  duplicate  factors.  A  ratio  of  8:1 
would  be  expected  if  the  two  genes  were  linked  with  crossing  over 
of   approximately  34  per   cent.      The   same  two   factors   in   the 


ENDOSPERM    CHARACTERS    IN    MAIZE  599 

repulsion  phase  should  give  a  ratio  of  17:1.  Ear  No.  2178  may 
represent  the  repulsion  phase  since  it  deviates  from  the  expected 
17:1  ratio  by  less  than  three  times  the  probable  error. 

It  is  also  possible  that  three  factors  instead  of  two  are  involved. 
Plants  heterozygous  for  two  of  these  and  homozygous  for  the 
third  would  be  expected  to  give  8:1  ratios  if  the  two  heterozy- 
gous factors  were  linked  with  crossing  over  of  34  per  cent. 
Plants  heterozygous  for  all  three,  two  of  which  are  linked,  should 
give  35:1  instead  of  63:1  ratios.  Ear  No.  2178  fits  such  a  ratio 
very  closely.  The  data,  however,  are  not  sufficiently  comprehen- 
sive to  substantiate  such  an  interpretation.  If  three  factors  are 
involved  with  34  per  cent  crossing  over  between  two  of  them,  the 
following  ratios  are  expected  when  dormant  seeds  from  ear  No. 
2178  are  grown;  3:1,  8:1,  15:1,  17:1,  35:1,  and  71:1.  Another 
season's  results  should  show  definitely  whether  duplicate  or  tripli- 
cate factors  are  involved. 

Premature  germination  in  this  stock  is  similar  to  that  caused 
by  Sei-  Germination  begins  at  a  very  late  stage  and  the  endo- 
sperm color  is  not  greatly  affected. 

SUMMARY  OF  BREEDING  BEHAVIOR 

Tentatively,  then,  the  genetic  situation  with  regard  to  the  stocks 
which  have  been  studied  may  be  outlined  as  follows : 

ge, 

ge.j,  >Five  single  factors,  any  one  of  which  in  a  homozygous 

ge±   recessive  condition  causes  premature  germination. 

ae  ~\  A  pair  of  independent  duplicate  factors  which  cause  pre- 
ge\  mature  germination  when  both  are  present  in  the  reces- 
sive condition 

ae  \  A  pair  of  linked  duplicate  factors  causing  premature  ger- 
%e*  f  urination  when  both  are  present.     Crossing  over  is  about 
6  °->  34  per  cent.      A  third   factor  in  this  set,  ge10,  is  also 
indicated. 

LINKAGE   RELATIONS 

A  detailed  study  of  the  linkage  relations  of  these  factors  with 
other  well  known'characters  has  not  yet  been  undertaken.  Data 
are  available,  however,  to  show  the  relation  of  the  gelt  ge2,  ges, 
and  ge5  genes  with  the  factor  for  sugary  endosperm.  In  all 
these  cases  the  germinating  seeds  cannot  be  accurately  classified 
with  regard  to  endosperm  texture  and  linkage  must  be  detected 
by  the  distortion  in  the  starchy  :  sugary  ratio  among  the  dormant 
seeds.     In  the  repulsion  phase  the  percentage  of  sugary  seeds  in 


600  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

the  dormant  class  should  vary  between  25  and  33^3,  depending-  on 
the  intensity  of  the  linkage.  This  is  shown  diagrammatically  in 
Figure  63,  Part  I.  In  the  coupling  phase,  linkage  would  be 
expected  to  cause  a  deficiency  of  sugary  seeds  in  the  dormant  class, 
the  percentage  ranging  from  25  to  o. 

gex  x  Su 

The  linkage  relations  of  gex  with  Su  are  determined  from  the 
cross  between  the  ge1  and  ge2  stocks.  The  former  is  a  yellow 
flint  variety,  the  latter  a  yellow  sugary.  Eight  ears  which  were 
segregating  for  ge1  were  obtained  from  this  cross  and  the  propor- 
tion of  sugary  seeds  in  the  dormant  class  on  these  ears  is  shown 
in  Table  27. 

gregation    in    Dormant    Seeds    from    Ears 
Segregating  gei. 


Table  27.  Starchy 

Sugary  Seg 

Seg 

Ear  No. 

Starchy 

710 

145 

715 

159 

721 

179 

722 

146 

723 

162 

725 

110 

726 

164 

727 

153 

Total 

I2l8 

Ex.  3:1 

1269 

Dev.  51 

P.  E.  12.0 

Sugary 

Percent  Sugary 

60 

29-3 

6l 

27.7 

73 

29.O 

69 

32.1 

44 

2I.4 

43 

28.1 

71 

30.2 

53 

25-7 

474 

28.0 

423 

25.0 

It  is  noted  that  there  is  an  excess  of  sugary  seeds,  amounting 
to  4.25  times  the  probable  error.  Such  a  deviation  would  be 
expected  by  chance  alone  only  once  in  about  250  trials.  The 
excess,  though  it  occurs  in  all  but  one  of  the  eight  ears,  would  not 
be  regarded  as  significant  if  seeds  from  a  smaller  number  of  ears 
were  counted.  When  all  the  ears  are  combined,  however,  the 
accumulation  of  small  deviations  is  one  direction  results  in  a 
total  deviation  which  can  scarcely  be  attributed  to  chance.  The 
excess  of  sugary  seeds  in  this  class  can  be  explained  by  assuming 
linkage  between  the  genes  ge1  and  Su  with  approximately  40 
per  cent  crossing  over.  It  is  realized,  of  course,  that  linkage 
values  determined  by  the  distortion  in  a  single  class  necessarily 
have  a  large  probable  error  and  the  value  given  must  not  be 
regarded  as  more  than  an  approximation. 

That  the  excess  of  sugary  seeds  in  the  dormant  class  is  due  to 
some  relation  with  the  gex  factor  and  not  to  errors  in  classifica- 
tion or  to  genetic  factors  affecting  the  rate  of  pollen  tube  growth, 
is  further  indicated  by  a  count  of  the  sugary  seeds  on  eight  ears 


ENDOSPERM    CHARACTERS    IN    MAIZE 


6oi 


from  the  same  cross  which  are  not  segregating'  for  gex.  On  these 
ears  the  percentage  of  sugary  seeds  is  very  close  to  expectation 
and  deviations  are  minus  almost  as  frequently  as  plus,  as  is  indi- 
cated in  Tahle  28. 

Table  28.     Starchy  :Sugary    Segregation    in    Ears    Not    Segregating    gei 
from  Same  Cross  as  Ears  in  Table  27. 


Ear  No. 

Starchy 

Sugary 

Percent  Sugary 

728 

192 

48 

20.0 

729 

221 

80 

26.6 

730 

211 

65 

23.6 

731 

204 

69 

25-3 

733 

163 

57 

25-9 

734 

185 

82 

30.7 

735 

227 

68 

23.0 

736 

175 

79 

3i. 1 

Total 

1578 

548 

25.8 

Ex.  3:1 

1594 

532 

25.0 

Dev.  16 

P.  E.  13.5 

ge2 

X  su 

The  relation  of  ge2  and  su  is  shown  by  ears  from  this  same 
cross  which  are  segregating  for  ge2  and  sugary  but  not  for  gex. 
The  counts  from  seven  such  ears  appear  in  Table  29.  Linkage 
in  this  case  would  be  indicated  by  a  deficiency  of  sugary  seeds 
since  the  two  factors  entered  the  cross  in  the  coupling  phase. 
Actually  there  is  a  slight  but  not  significant  excess  of  sugary 
seeds  in  the  dormant  class  and  it  appears  safe  to  conclude  that 
ge2  and  su  are  independent. 


Table  29.     Starchy :  Sugary     Segregation     in     Dormant     Seeds     of 
Segregating  gei  from  Same  Cross  as  Ears  in  Tables  27  and  28. 


Ears 


Ear  No. 

Starchy 

705 
706 
707 
708 

121 
172 
I46 
158 

716 

134 

717 

155 

718 

152 

Total 
Ex.  3:1 
Dev.  26 

IO38 
IO64 

P.  E.  1 1.0 

Sugary 

Percent  Sugary 

44 

26.7 

59 

25-5 

67 

3i.5 

52 

24.8 

4i 

23-4 

54 

25.8 

64 

29.6 

38i 

26.8 

355 

25.0 

ge3  and  ge5  x  su 

Five  of  the  ears  represented  in  Table  24  are  segregating  for 
su,  ge3  and  ge5.  The  two  types  of  germinating  seeds  cannot  be 
distinguished  but  if  either  one  is  linked  with  sugary  a  distortion 


602  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

of  the  starchy  :  sugary  ratio  in  the  dormant  seeds  would  be 
expected.  A  count  of  the  two  types  of  seed  in  the  dormant  class 
on  these  five  ears  appears  in  Table  30.  The  agreement  with 
expectation  on  the  basis  of  independent  inheritance  is  very  good 
and  it  seems  certain  that  both  factors  are  independent  of  sugary. 

Table  30.     Starchy :  Sugary     Segregation     in     Dormant     Seeds     of     Ears 
Segregating  ge3  and  ges. 

Ear  No.  Starchy  Sugary  Percent  Sugary 

2039  99                       23                       18.9 

2041  121                       44                       26.7 

2043  96  35  26-7 

2044  154  58  27.4 

2046  99  29  22.7 

2047  124  47  27.5 

Total  693  236  25.4 

Ex.  3:1  697  232  25.0 

Dev.  4 
P.  E.  8.9 

APPARENT  LINKAGE  WITH  ENDOSPERM  COLOR  FACTORS 

As  has  already  been  mentioned,  there  is  a  strong  association 
between  germinating  seeds  and  absence  of  color  in  the  endosperm. 
In  the  case  of  ger,  gez  and  ge5  the  recessive  seeds  are,  with  few 
exceptions,  completely  white,  while  in  the  ge2  strain  the  germinat- 
ing seeds  are  a  pale  yellow. 

Table  31.     Apparent  Linkage  between  gei  and  White  Endosperm. 
, Yellow s  , White N 


Ear  No. 

Ge 

ge 

Ge 

ge 

710 

204. 

I 

I 

66 

715 

219 

0 

I 

56 

721 

252 

I 

0 

67 

722 

213 

I 

2 

74 

723 

205 

0 

I 

62 

726 

234 

0 

I 

61 

754 

205 

0 

I 

74 

Total  1532  3  7  460 

Occasionally,  however,  germinating  seeds  with  yellow  endo- 
sperm are  found  as  well  as  white  seeds  which  have  not  sprouted. 
The  frequency  of  these  exceptions  in  stocks  1  and  3  is  shown  in 
Tables  31  and  32.  At  first  glance  the  situation  represents  a  clear 
cut  case  of  close  linkage  with  less  than  1  per  cent  of  crossing 
over.  Eyster  (1924)  has  assumed  this  to  be  the  situation  in  his 
stock  and  has  calculated  the  amount  of  crossing  over  as  1.26  per 
cent.  The  writer  (1923)  had  previously  suggested  physiological 
correlation  as  an  explanation  of  these  results  and  the  evidence 


ENDOSPERM    CHARACTERS    IN    MAIZE  603 

indicates  that  this  is  probably  the  correct  interpretation  in  the 
stocks  reported  here. 

There  are  at  least  three  series  of  facts  which  are  not  compatible 
with  a  linkage  hypothesis  : 

1.  All  of  the  stocks  in  which  the  association  between  pre- 
mature germination  and  color  of  endosperm  appears  have 
originated  from  varieties  which  were  homozygous  for  yellow 
endosperm.  White  seeds  might  be  expected  to  arise  occasionally 
by  mutation,  but  the  appearance  of  four  genetically  distinct  factors 
for  white  seeds,  each  one  closely  linked  with  a  factor  for  germi- 
nating seeds,  cannot  reasonably  be  assumed. 

2.  When  pollen  from  plants  which  are  segregating  for  germi- 
nating seeds  is  applied  to  silks  of  a  white  variety,  only  yellow 
seeds  are  produced.  If  the  segregating  plants  were  heterozy- 
gous for  endosperm  color,  as  they  appear  to  be,  such  crosses 
should  produce  I  :i  ratios,  providing  that  the  white  endosperm  of 
the  germinating  seeds  has  the  same  genetic  basis  as  the  white 
endosperm  of  common  white  varieties. 

3.  The  apparent  cross  overs,  white  seeds  which  fail  to  sprout, 
should  breed  true  if  it  is  assumed  that  they  are  homozygous  for 
a  recessive  endosperm  color  factor.  Only  a  small  number  of  these 
seeds  have  been  available  but  all  those  which  were  grown  pro- 
duced only  plants  segregating  for  white  seeds  which  germinated, 
with  few  exceptions. 

Table  32.    Apparent  Linkage  between  ge3  and  White  Endosperm. 

, Yellow -,  , White , 

Ear  No.  Ge  ge  Ge  ge 


758 

70 

I 

0 

22 

760 

60 

0 

0 

17 

761 

IOI 

0 

0 

27 

Total  231  1  o  66 

If  the  association  between  germinating  seeds  and  endosperm 
color  is  not  due  to  linkage,  to  what  may  it  be  attributed  ?  A  histo- 
logical study  of  the  germinating  seeds  of  the  ge1  stock  has  given 
evidence  which  seems  to  have  some  bearing  on  this  question. 

PREMATURE  DIGESTION  AND  PIGMENT  FORMATION 

Some  of  the  white  seeds  were  removed  from  the  ear  at  an  early 
stage,  killed  and  fixed,  imbedded  in  paraffin,  sectioned,  and 
stained.  It  was  found  that  even  at  this  early  stage  the  processes 
of  germination  had  already  begun.  The  cells  in  the  epithelial 
layer  of  the  scutellum  had  elongated  and  the  invaginations  of  this 
layer,  so  characteristic  of  mature  seeds  of  maize,  were  already 
apparent.     Sargant  and  Robertson  (1905)  have  made  a  thorough 


604  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

study  of  the  scutellum  of  maize  and  are  of  the  opinion  that  these 
invaginations  are  glandular  in  nature  and  that  their  function  is 
the  secretion  of  diastase.  There  is  some  appearance  of  digestion 
in  the  cells  of  the  endosperm  of  germinating  seeds  even  at  the 
early  milk  stage,  and  in  material  gathered  from  the  same  ears  a 
week  later,  the  digestion  is  quite  marked. 

It  is  possible  that  the  normal  production  of  color  in  the  cells  of 
the  endosperm  cannot  proceed  while  digestion  is  occurring  in 
these  cells.  The  yellow  color  in  the  endosperm  is  found  in  the 
matrix  which  surround  the  starch  grains,  and  if  this  matrix  is 
being  digested  as  rapidly,  or  more  rapidly,  than  new  material  is 
being  supplied  by  the  plant,  it  is  hardly  to  be  expected  that  pig- 
ment formation  would  proceed  in  the  normal  fashion. 

The  apparent  cross  overs,  the  yellow  seeds  which  germinate  and 
the  white  seeds  which  remain  dormant,  may  be  merely  variations 
of  this  condition. 

Some  of  the  germinating  seeds  probably  remain  yellow  because 
the  digestion  does  not  begin  soon  enough  or  is  not  rapid  enough 
to  inhibit  the  formation  of  endosperm  color.  This  would  appear 
to  be  the  case  in  the  stock  where  the  duplicate  factors  ge6  and  ge- 
are  involved.  In  this  stock  the  pale  yellow  is  usually  confined  to 
an  area  adjacent  to  the  embryo  and  the  dorsal  side  of  the  seed 
retains  the  full  yellow  color. 

The  other  class  of  apparent  cross  overs,  the  white  dormant 
seeds,  are  more  difficult  to  explain.  The  fact  that  all  of  these 
seeds  which  have  ever  been  grown  have  given  ears  segregating 
for  germinating  seeds  might  suggest  that  this  character  occa- 
sionally manifests  itself  in  the  heterozygous  condition. 

THE  RELATION  OE  PREMATURE  GERMINATION   TO 
CHLOROPHYLL    DEVELOPMENT 

Evidently  there  is  also  a  physiological  relation  of  some  sort 
between  premature  germination  and  chlorophyll  development. 
Types  which  begin  germination  at  a  very  early  stage,  such  as  ge~ 
and  ge5  always  produce  pure  white  sprouts.  The  ge1  type,  in 
which  germination  begins  somewhat  later,  ordinarily  produces 
white  plumules  but  occasionally  these  show  a  tinge  of  green. 
The  remaining  types  in  which  germination  begins  only  after  the 
kernels  are  well  developed,  produce  only  normal  green  sprouts. 
Apparently  the  premature  germination,  if  it  begins  early  enough, 
completely  inhibits  the  formation  of  chorophyll  just  as  it  pre- 
vents the  laying  down  of  yellow  pigment  in  the  endosperm. 

This  association,  too,  is  characterized  by  occasional  exceptions. 
Germinating  seed  of  ge1}  ge3  and  ge5  are  sometimes  found  which 
produce  sprouts  of  normal  green  color,  but  dormant  seeds  which 
Sfive  albino  seedlings  when  germinated  have  never  been  observed. 


ENDOSPERM    CHARACTERS    IN    MAIZE  605 

PHYSIOLOGY    OF    PREMATURE    GERMINATION 

Oppenheimer  (1922)  has  found  that  in  seeds  of  tomato,  gourd, 
cucumber  and  Nicotiana  rustica,  germination  can  be  suppressed 
by  surrounding  the  seeds  with  crushed  tissue  of  the  receptacles 
of  the  mother  plants  or  by  growing  them  on  filter  paper  saturated 
with  an  extract  from  these  tissues. 

The  degree  of  suppression  is  approximately  proportional  to  the 
amount  of  tissue  present  or  the  concentration  of  the  extract. 
This  suppression  can  be  overcome  by  heating  the  tissue  or  extract 
to  ioo°  C.  Apparently  the  mother  plants  of  these  species  nor- 
mally supply  the  growing  seed  with  inhibiting  substances  which 
prevent  germination  while  the  seeds  are  still  attached  to  the  plant. 
Maze  (1910)  is  of  the  opinion,  and  presents  some  evidence  in 
favor  of  his  view,  that  dormancy  in  seeds,  buds,  bulbs,  and  tubers 
is  due,  in  some  cases,  to  the  action  of  volatile  esters  which  prevent 
growth  until  they  are  eliminated. 

Oppenheimer  did  not  include  seeds  of  maize  in  his  experiments 
but  a  test  made  by  the  writer,  in  an  effort  to  determine  at  what 
stage  of  development  germination  in  the  normal  seed  still  attached 
to  the  plant  could  be  induced,  may  have  some  bearing  on  the 
problem.  An  ear  in  the  early  milk  stage  was  stripped  down  and 
wrapped  with  cotton.  Around  this  were  wrapped  several  layers 
of  cloth.  The  ends  of  the  cloth  were  submerged  in  a  vessel  of 
water  and  served  as  a  wick,  keeping  the  cotton  surrounding  the 
ear  constantly  saturated.  The  grains  swelled  considerably,  indi- 
cating that  water  was  being  absorbed,  but  no  germination 
occurred.  A  number  of  seeds  which  had  been  removed  from  this 
ear  and  placed  in  an  ordinary  germinator  at  approximately  the 
same  temperature  and  with  the  same  moisture  supply,  began  to 
sprout  after  about  ten  days.  This  is  much  longer  than  the  time 
required  by  immature,  dry  seeds  to  germinate  under  the  same  con- 
ditions and  indicates  that  inhibiting  substances  were  first  elimi- 
nated before  germination  could  begin. 

THE  EFFECTS  OF  PREMATURE  GERMINATION  ON  THE 
GROWTH    OF  THE   SEED 

An  attempt  was  made  to  determine  whether  the  germinating 
seeds  receive  the  normal  amount  of  nourishment  from  the  plant 
while  germination  is  going  on  or  whether  these  seeds  cease  their 
development  after  germination  begins.  Ears  which  were  segre- 
gating for  germinating  seeds  (get)  were  harvested  at  three  weeks 
after  pollination,  and  at  intervals  of  one  week  thereafter,  until 
maturity.  These  ears  were  dried  on  a  rack  until  thoroughly  dry, 
at  which  time  the  kernels  were  shelled  off,  the  dormant  and  ger- 
minating seeds  separated,  counted  and  weighed,  and  the  average 
weight  of  each  class  determined  by  simple  division.     The  results 


6o6 


CONNECTICUT    EXPERIMENT    STATION 


BULLETIN    279 


are  shown  in  two  curves  in  Fig.  76.  It  will  be  seen  that  already 
in  the  early  milk  stage  there  was  a  noticeable  difference  in  the 
relative  development  of  the  two  types  as  represented  by  their  dry 
weights.  This  difference  increased  in  the  second  week  under 
observation  and  thereafter  the  germinating  seeds  no  longer  in- 
creased in  weight  and  actually  fell  off  somewhat  during  the  last 


400 


^     300 

a 

SZ5 


C5 
H 


I 


200 


100 


i 

L-— -~~~~~ """ """"" 

lA 

^r        , 

1 

iB 

WEEKS  AFTER  POLLINATION 

Fig.  76. — Growth  curves  of  dormant  and  germinating  (gci)  seeds  from 
the  same  segregating  ears.  The  sprouting  seeds  do  not  receive  enough 
nourishment  from  the  plant  to  replace  the  material  lost  in  germination. 


three  weeks.  The  normal  seeds  on  the  same  ears  showed  an 
increase  in  dry  matter  during  every  week  of  the  test.  It  is  evident 
from  these  two  growth  curves  that  the  amount  of  nourishment 
supplied  to  the  aberrant  seeds  by  the  plant  is  not  sufficient  to 
replace  that  consumed  in  germination.  In  fact,  it  is  quite  likely 
that  the  germinating  seeds  become  partially  or  wholly  "physi- 
ologically isolated"  from  the  plant  during  the  later  periods.  At 
maturity  the  germinated  seeds  weighed  only  51  per  cent  as  much 
as  the  dormant  seeds  from  the  same  ears. 


ENDOSPERM    CHARACTERS    IN    MAIZE  607 

DISCUSSION 

For  a  period  of  several  weeks,  while  the  seed  is  in  the  milk  or 
dough  stage,  natural  conditions  for  germination  are  almost  at  an 
optimum.  The  temperature  is  fairly  high  and  the  moisture 
supply  is  abundant.  The  embryos  are  sufficiently  developed  to 
produce  plants  capable  of  surviving  and  the  endosperm  contains 
enough  food  material  to  nourish  the  seedling  until  it  begins  to 
manufacture  food  for  itself.  That  this  is  true  is  shown  by  the 
behavior  of  immature  seeds  harvested  at  these  early  stages  when 
many  of  them  are  capable  not  only  of  germinating  but  of  produc- 
ing almost  a  normal  yield  of  grain.      (See  Part  I.) 

Why  is  it,  then,  that  the  partially  developed  seed  ordinarily 
never  germinates  while  still  attached  to  the  plant?  Apparently 
the  mother  plant,  though  it  provides  conditions  suitable  for  ger- 
mination, at  the  same  time  supplies  inhibiting  substances  which 
prevent  germination  from  beginning. 

The  physiological  processes  involved  in  maintaining  a  period  of 
dormancy,  which  permits  the  embryo  to  attain  a  maximum  devel- 
opment and  the  endosperm  to  accumulate  a  mass  of  food  material, 
are  probably  very  complicated.  It  is  not  at  all  surprising,  there- 
fore, to  find  a  number  of  distinct  genetic  factors  operating  during 
this  period.  Every  stage  in  the  ontogeny  of  the  sporophyte  is 
evidently  controlled  by  many  genetic  factors  and  the  maintenance 
of  a  normal  period  of  dormancy  which  prevents  premature  ger- 
mination with  its  disastrous  effects,  and  permits  the  sporophyte 
to  pass  safely  through  unfavorable  seasons,  is  no  exception. 
Nine  Mendelian  factors  which  govern  this  stage  have  already 
been  identified.  Many  others  will  undoubtedly  be  found  as  maize 
is  investigated  more  extensively. 

Summary — Part  IV 

1.  Nine  Mendelian  factors  involved  in  the  maintenance  of  a 
normal  period  of  dormancy  in  maize  seeds  have  been  identified. 

2.  Five  of  these  are  complementary  factors.  When  any  one 
of  these  is  lacking  the  seed  germinates  prematurely.  Plants 
heterozygous  for  one,  two  or  three  factors  give  3:1,  9:7,  and 
27 :37  ratios  respectively. 

3.  A  pair  of  independent  duplicate  factors  results  in  ratios 
of  15  dormant:  1  germinating  when  plants  are  heterozygous  for 
both. 

4.  A  pair  of  linked  duplicate  factors  gives  8  :i  ratios  when 
plants  are  heterozygous  for  both.  Crossing  over  is  about  34 
per  cent. 

5.  The  gex  factor  appears  to  be  linked  with  the  gene  for  sugary 
endosperm.  Crossing  over  is  about  40  per  cent.  ge2,  gez  and 
ge5  are  found  to  be  independent  of  sugary. 


608  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    279 

6.  An  apparent  case  of  close  linkage  between  endosperm  color 
and  several  types  of  germinating  seeds  is  probably  due  to  the 
physiological  effects  of  premature  germination  upon  the  accumula- 
tion of  pigment  in  the  cells  of  the  endosperm. 

7.  A  similar  association  between  germinating  seeds  and  white 
seedlings  may  also  be  due  to  physiological  complications.  Seeds 
which  germinate  at  early  stage  produce  only  white  plumules ; 
those  which  germinate  later  have  normal  green  sprouts. 

8.  Premature  germination  is  apparently  caused  by  the  lack  or 
loss  of  inhibiting  substances  normally  supplied  by  the  plant  to 
the  growing  seeds. 

9.  It  is  suggested  that  many  genetic  factors  are  involved  in  the 
maintenance  of  a  normal  period  of  dormancy  in  maize  seeds. 


Conclusion 

The  mature,  dormant  seed  of  maize  with  its  well  developed 
embryo  and  the  cells  of  its  endosperm  packed  with  starch  grains, 
represents  a  real  organic  achievement. 

Each  ovule  has  its  separate  style ;  each  style,  in  order  for  a 
seed  to  develop,  must  receive  a  pollen  grain  capable  of  germinat- 
ing and  producing  a  tube  sufficiently  vigorous  to  reach  the  micro- 
pyle.  Failure  of  the  growing  tube  to  attain  its  goal  results  in  the 
production  of  "parthenocarpic"  defectives  without  endosperm  or 
embryo. 

After  the  pollen  tube  has  entered  the  micropyle,  a  very  precise 
mechanism  of  fertilization  begins  to  function.  Failure  of  this 
intricate  mechanism  in  any  detail  may  cause  the  formation  of 
"germless"  seeds,  lacking  an  embryo,  "miniature"  seeds  in  which 
the  endosperm  is  greatly  reduced  in  size  or  perhaps  aborted  seeds 
of  several  other  types. 

The  fertilization  mechanism  having  functioned  properly,  the 
growing  seed  begins  to  receive  the  influence  of  various  genetic 
factors.  Thirteen  distinct  factors  have  been  found  which  arrest 
the  development  of  the  seed  and  cause  it  to  be  defective  and  in- 
capable of  normal  growth  and  germination.  Five  additional 
factors  may  affect  the  nature  of  the  stored  food  material  to  such 
a  degree  that  the  seed  is  handicapped  and  cannot  attain  a  maximum 
development. 

In  addition  to  the  18  genetic  factors  so  far  found  which  retard 
development  to  a  greater  or  lesser  degree,  nine  other  factors  have 
appeared  which  stimulate  certain  functions  prematurely,  with 
equally  disastrous  consequences.  The  seed,  in  order  to  reach 
maturity  and  pass  safely  through  unfavorable  seasons  must 
remain  dormant  while  still  attached  to  the  plant,  even  though  it 
is  capable  of  germination  at  this  stage  and  the  conditions  favor- 
ing germination  are  almost  optimum.     Five  complementary  factors 


ENDOSPERM    CHARACTERS    IN    MAIZE  609 

and  two  pairs  of  duplicate  factors  are  involved  in  the  mainten- 
ance of  dormancy  during  development.  The  loss  of  any  one  or 
pair  of  these  causes  the  seed  to  germinate  prematurely  with  fatal 
results. 

A  fully  mature,  normally  developed,  dormant,  white,  starchy 
seed,  then,  represents  the  cumulative  action  of  27  Mendelian 
factors  of  which  we  know  the  mode  of  inheritance.  How  many 
additional  factors  are  involved  would  be  difficult  to  estimate,  but 
since  all  these  permanent  departures  from  the  normal  condition  of 
the  germplasm  have  been  found  in  a  limited  amount  of  material, 
it  is  certain  that  many  more  hereditary  factors  of  a  similar  nature 
will  appear.  This  gives  some  clue  as  to  the  infinitely  large  num- 
ber of  genes  always  working  to  produce  a  normal  seed.  The 
majority  of  these  can  not  be  known  because  they  do  not  vary. 

All  hereditary  units  here  studied  concern  only  the  seed  which 
comprises  a  brief  period  betweeen  fertilization  and  the  resting 
stage  of  the  embryo.  What,  therefore,  must  be  involved  in  the 
ontogeny  of  the  entire  plant?  The  young  seedling,  the  growing 
plant,  the  chlorophyll  processes,  reproductive  machinery  and  even 
the  gametophyte  generation  are  all  controlled  by  genetic  factors 
the  tabulation  of  which  has  onlv  been  started. 


6lO  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    2/9 

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6 14  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    2~9 


EXPLANATION    OF    PLATES. 

Plate  XXI.     Longitudinal  sections  of  normal  seed  and  three  successive 
stages  of  development  of  defective  seeds  of  den  stock,     x  n. 

1.  Normal  seed  at  early  milk  stage. 

2.  Defective  seed,  early  milk.* 

3.  Defective  seed,  late  milk. 

4.  Defective  seed,  dough. 

Plate  XXII.     Three  successive  stages  in  development  of  defective  seeds 
of  the  des  stock,     x  11. 

1.  Defective  seed,  early  milk. 

2.  Defective  seed,  late  milk. 

3.  Defective  seed,  dough. 

Plate  XXIII.     Three  successive  stages  in  development  of  defective  seeds 
of  the  des  stock,     x  11. 

1.  Defective  seed,  early  milk. 

2.  Defective  seed,  late  milk. 

3.  Defective  seed,  early  dough. 

Plate    XXIV.     Successive    stages    in    the    development    of    the    normal 


embryo 


x  15. 

Blister  stage. 
Early  milk. 
Milk. 

Late  milk. 
Dough. 


Plate  XXV.  1.  Ears  of  a  uniform  first  generation  hybrid  harvested 
at  successive  stages  of  maturity.  From  left  to  right  the  ears  represent 
stages  of  14,  21,  28,  35,  41,  51  and  75  days  after  pollination. 

2.  Fifty  seeds  from  each  of  the  ears.  In  appearance  and  dry  weight 
these  normal  seeds  harvested  at  successive  stages  of  development  resemble 
various  types  of  hereditary  defectives. 

3.  The  results  of  planting  the  fifty  seeds  shown  in  2.  In  ability  to 
germinate  the  immature  normal  seeds  are  superior  to  hereditary  defectives 
of  the  same  relative  development. 

Plate  XXVI.  Four  morphologically  distinct  types  of  seeds  which  may 
occur  on  any  ear  of  maize,     x  11. 

1.  Normal  seed  with  well  developed  endosperm  and  embryo. 

2.  Hereditary  defective  with  aborted  endosperm  and  embryo. 

3.  Germless  seed  containing  endosperm  but  no  embryo.     This  type 

is  probably  due  to  single  fertilization. 

4.  Parthenocarpic    defective    with    neither    endosperm    nor    embryo. 

This  type   is   caused  by  pollination   which   fails   to  accomplish 
fertilization. 


*  The  relative  stage  of  development,  in  every  case,  is  that  of  normal  seeds 
from  the  same,  ear,  and  not  of  the  defectives  themselves. 


PLATE  XXI. 


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PLATE  XXII. 


PLATE  XXIII. 


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PLATE  XXIV, 


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PLATE  XXV 


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PLATE  XXVI. 


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